Structural basis for the functional and inhibitory mechanisms of β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) of Plasmodium falciparum

The β-hydroxyacyl-acyl carrier protein dehydratase of Plasmodium falciparum (PfFabZ) catalyzes the third and important reaction of the fatty acid elongation cycle. The crystal structure of PfFabZ is available in hexameric (active) and dimeric (inactive) forms. However, PfFabZ has not been crystallized with any bound inhibitors until now. We have designed a new condition to crystallize PfFabZ with its inhibitors bound in the active site, and determined the crystal structures of four of these complexes. This is the first report on any FabZ enzyme with active site inhibitors that interact directly with the catalytic residues. Inhibitor binding not only stabilized the substrate binding loop but also revealed that the substrate binding tunnel has an overall shape of “U”. In the crystal structures, residue Phe169 located in the middle of the tunnel was found to be in two different conformations, open and closed. Thus, Phe169, merely by changing its side chain conformation, appears to be controlling the length of the tunnel to make it suitable for accommodating longer substrates. The volume of the substrate binding tunnel is determined by the sequence as well as by the conformation of the substrate binding loop region and varies between organisms for accommodating fatty acids of different chain lengths. This report on the crystal structures of the complexes of PfFabZ provides the structural basis of the inhibitory mechanism of the enzyme that could be used to improve the potency of inhibitors against an important component of fatty acid synthesis common to many infectious organisms.     

EBP2 plays a key role in Epstein-Barr virus mitotic segregation and is regulated by aurora family kinases

Epstein-Barr virus (EBV) genomes persist indefinitely in latently infected human cells, in part due to their ability to stably segregate during cell division. This process is mediated by the viral EBNA1 protein, which tethers the viral episomes to the cellular mitotic chromosomes. We have previously identified a mitotic chromosomal protein, human EBNA1 binding protein 2 (hEBP2), which binds to EBNA1 and enables EBNA1 to partition EBV-based plasmids in Saccharomyces cerevisiae. Using an RNA silencing approach, we show that hEBP2 is essential for the proliferation of human cells and that repression of hEBP2 severely decreases the ability of EBNA1 and EBV-based plasmids to bind mitotic chromosomes. When expressed in yeast, hEBP2 undergoes the same cell cycle-regulated association with the mitotic chromatin as in human cells, and using yeast temperature-sensitive mutant strains, we found that the attachment of hEBP2 to mitotic chromosomes was dependent on the Ipl1 kinase. Both RNA silencing of the Ipl1 orthologue in human cells (Aurora B) and specific inhibition of the Aurora B kinase activity with a small molecule confirmed a role for this kinase in enabling hEBP2 binding to human mitotic chromosomes, suggesting that this kinase can regulate EBV segregation.     

Differential apoptotic and redox regulatory activities of curcumin and its derivatives

We have synthesized different bioconjugates of curcumin, which were tested for their pro- and antioxidant properties. In the present study five representative derivatives of curcumin, i.e., 4,4'-di-(O-acetyl) curcumin, 4,4'-di-(O-glycinoyl) curcumin, 4,4'-di-(O-glycinoyl-di-N-piperoyl) curcumin, 4,4'-di-(O-piperoyl) curcumin, and 4,4'-(O,O-cystinoyl)-3,3'-dimethoxydiphenyl-1,6-heptadiene-3,5-dione, were used for testing their apoptotic potential on tumor cells. Dipiperoyl and diglycinoyl derivatives showed higher apoptotic activity at lower concentrations, whereas diacetyl curcumin had slightly lower apoptotic activity on tumor cells. On the other hand, diglycinoyl-dipiperoyl and cystinoyl heptadiene derivatives had lost their apoptotic potential significantly. The apoptotic activity of these derivatives correlated very well with the generation of ROS by the tumor cells, whereas GSH levels remained unaltered. Our studies also indicate downregulation of Bcl-2 and participation of caspase-3 in the apoptotic death of tumor cells.     

Improving the yield and quality of DNA isolated from white-rot fungi

A new simple method used to eliminate polysaccharides that cause problems during DNA isolation was established for 6 different white-rot fungi using 1% hexadecyltrimethylammonium bromide (CTAB) as wash buffer and followed by centrifugation. Variation in the DNA yield and quality was ascertained using precipitating agents, detergents and cell-wall-hydrolyzing chitinase. Considerable amount of exopolysaccharides from fungal biomass was removed with the use of 1% CTAB wash buffer followed by centrifugation. The DNA varied in terms of yield and quality. For the DNA extraction use of 2% SDS in extraction buffer worked best for Pycnoporus cinnabarinus, Cyathus bulleri, Cyathus striatus and Cyathus stercoreus, while 2% CTAB worked best for Phanerochaete chrysosporium and Pleurotus ostreatus. Elimination of phenol and use of absolute ethanol for precipitating DNA resulted in good yield and quality of DNA. This DNA was amenable to restriction endonuclease digestion.     

Mechanistic analysis of the mitotic kinesin Eg5

Eg5 is a slow, plus-end-directed microtubule-based motor of the BimC kinesin family that is essential for bipolar spindle formation during eukaryotic cell division. We have analyzed two human Eg5/KSP motors, Eg5-367 and Eg5-437, and both are monomeric based on results from sedimentation velocity and sedimentation equilibrium centrifugation as well as analytical gel filtration. The steady-state parameters were: for Eg5-367: k(cat) = 5.5 s(-1), K(1/2,Mt) = 0.7 microm, and K(m,ATP) = 25 microm; and for Eg5-437: k(cat) = 2.9 s(-1), K(1/2,Mt) = 4.5 microm, and K(m,ATP) = 19 microm. 2'(3')-O-(N-Methylanthraniloyl)-ATP (mantATP) binding was rapid at 2-3 microm(-1)s(-1), followed immediately by ATP hydrolysis at 15 s(-1). ATP-dependent Mt.Eg5 dissociation was relatively slow and rate-limiting at 8 s(-1) with mantADP release at 40 s(-1). Surprisingly, Eg5-367 binds microtubules more effectively (11 microm(-1)s(-1)) than Eg5-437 (0.7 microm(-1)s(-1)), consistent with the steady-state K(1/2,Mt) and the mantADP release K(1/2,Mt). These results indicate that the ATPase pathway for monomeric Eg5 is more similar to conventional kinesin than the spindle motors Ncd and Kar3, where ADP product release is rate-limiting for steady-state turnover.     

Imbalanced base excision repair in response to folate deficiency is accelerated by polymerase beta haploinsufficiency

The mechanism by which folate deficiency influences carcinogenesis is not well established, but a phenotype of DNA strand breaks, mutations, and chromosomal instability suggests an inability to repair DNA damage. To elucidate the mechanism by which folate deficiency influences carcinogenicity, we have analyzed the effect of folate deficiency on base excision repair (BER), the pathway responsible for repairing uracil in DNA. We observe an up-regulation in initiation of BER in liver of the folate-deficient mice, as evidenced by an increase in uracil DNA glycosylase protein (30%, p < 0.01) and activity (31%, p < 0.05). However, no up-regulation in either BER or its rate-determining enzyme, DNA polymerase beta (beta-pol) is observed in response to folate deficiency. Accordingly, an accumulation of repair intermediates in the form of DNA single strand breaks (37% increase, p < 0.03) is observed. These data indicate that folate deficiency alters the balance and coordination of BER by stimulating initiation without subsequently stimulating the completion of repair, resulting in a functional BER deficiency. In directly establishing that the inability to induce beta-pol and mount a BER response when folate is deficient is causative in the accumulation of toxic repair intermediates, beta-pol-haploinsufficient mice subjected to folate deficiency displayed additional increases in DNA single strand breaks (52% increase, p < 0.05) as well as accumulation in aldehydic DNA lesions (38% increase, p < 0.01). Since young beta-polhaploinsufficient mice do not spontaneously exhibit increased levels of these repair intermediates, these data demonstrate that folate deficiency and beta-pol haploinsufficiency interact to increase the accumulation of DNA damage. In addition to establishing a direct role for beta-pol in the phenotype expressed by folate deficiency, these data are also consistent with the concept that repair of uracil and abasic sites is more efficient than repair of oxidized bases.     

Abo blood groups and completed reproductive performance of rural Haryanavi couples: analysing measures of selection intensities

The possible differential effects of ABO blood group materno-paternal (fetal) incompatibility on completed reproductive performance were investigated on a sample of 100 couples (100 fathers and 100 mothers) from three villages in the Jind district of Haryana state, India. The average number of live births per mating couple was slightly higher for the incompatible matings (5.32) than the compatible ones (5.05). This advantage was offset by higher postnatal mortality in the former. Consequently, the average number of living children in the compatible matings (4.64) was higher than in the incompatible ones (4.18). With reference to individual ABO matings, the index of relative fertility (Irf) was the highest in A x AB followed by B x A type of incompatible matings. No decrease in live births in O x A and O x B incompatible matings was observed compared with their reciprocal compatible ones, i.e. A x O and B x O matings, as has been hypothesized in previous studies. The total pregnancy wastage was substantially higher in ABO-incompatible matings (24.59%) than compatible matings (8.45%). About 71% of the postnatal deaths took place within one year of the birth in the case of incompatible matings compared with 50% in the case of compatible matings. The study supports the hypothesis that selection is operative at the ABO locus as revealed by the measures of selection intensity. The loss of fitness in the present sample was associated with differential mortality. There were no differences in the proportions of average number of male live births in the compatible (0.55) and incompatible matings (0.58). However, in the individual mating types, there was some evidence of higher or lower proportions of male live births.