Purification, refolding and autoactivation of the recombinant cysteine proteinase EhCP112 from Entamoeba histolytica

The cysteine proteinase EhCP112 and the adhesin EhADH112 assemble to form the EhCPADH complex involved in Entamoeba histolytica virulence. To further characterize this cysteine proteinase, the recombinant full-length EhCP112 enzyme was expressed and purified under denaturing conditions. After a refolding step under reductive conditions, the inactive precursor (ppEhCP112) was processed to a 35.5 kDa mature and active enzyme (EhCP112). The thiol specific inhibitor E-64, but not serine or aspartic proteinase inhibitors arrested this activation process. The activation step of the proenzyme followed by the mature enzyme suggests an autocatalytic process during EhCP112 maturation. The experimentally determined processing sites observed during EhCP112 activation lie close to processing sites of other cysteine proteinases from parasites. The kinetic parameters of the mature EhCP112 were determined using hemoglobin and azocasein as substrates. The proteinase activity of EhCP112 was completely inhibited by thiol inhibitors, E-64, TLCK, and chymostatin, but not by general proteinase inhibitors. Since EhCP112 is a proteinase involved in the virulence of E. histolytica, a reliable source of active EhCP112 is a key step for its biochemical characterization and to carry out future protein structure-function studies.     

Molecular Design,Synthesis and Trypanocidal Activity of Dipeptidyl Nitriles as Cruzain Inhibitors

A series of compounds based on the dipeptidyl nitrile scaffold were synthesized and assayed for their inhibitory activity against the T. cruzi cysteine protease cruzain. Structure activity relationships (SARs) were established using three, eleven and twelve variations respectively at the P1, P2 and P3 positions. A K_i value of 16 nM was observed for the most potent of these inhibitors which reflects a degree of non-additivity in the SAR. An X-ray crystal structure was determined for the ligand-protein complex for the structural prototype for the series. Twenty three inhibitors were also evaluated for their anti-trypanosomal effects and an EC₅₀ value of 28 μM was observed for the most potent of these. Although there remains scope for further optimization, the knowledge gained from this study is also transferable to the design of cruzain inhibitors based on warheads other than nitrile as well as alternative scaffolds.     

Fuzziness and noise in nucleosomal architecture

Nucleosome organization plays a key role in the regulation of gene expression. However, despite the striking advances in the accuracy of nucleosome maps, there are still severe discrepancies on individual nucleosome positioning and how this influences gene regulation. The variability among nucleosome maps, which precludes the fine analysis of nucleosome positioning, might emerge from diverse sources. We have carefully inspected the extrinsic factors that may induce diversity by the comparison of microccocal nuclease (MNase)-Seq derived nucleosome maps generated under distinct conditions. Furthermore, we have also explored the variation originated from intrinsic nucleosome dynamics by generating additional maps derived from cell cycle synchronized and asynchronous yeast cultures. Taken together, our study has enabled us to measure the effect of noise in nucleosome occupancy and positioning and provides insights into the underlying determinants. Furthermore, we present a systematic approach that may guide the standardization of MNase-Seq experiments in order to generate reproducible genome-wide nucleosome patterns.     

Enhanced photosynthetic output via dichroic beam-sharing

Microbial solar biofuels offer great promise for future sustainable food, fuels and chemicals but are limited by low productivities and a requirement for large land areas to harvest sunlight. A 71?% increase in combined photosynthetic activity was achieved by illuminating both Rhodobacter sphaeroides and Arthrospira (Spirulina) platensis from a single beam of simulated sunlight, divided using a dichroic mirror. Therefore, this technique is termed ‘dichroic beam-sharing’, in which the complementary action spectra of two different useful micro-organisms, belonging to green and purple groups, is exploited and allows a single beam of sunlight to be shared efficiently between separate photobioreactors. Because the action spectra of these two organisms are typical of large groups, this novel method could increase the productivity of photosynthetic micro-organisms in the production of diverse commodities.     

Exploring polymorphisms in B-DNA helical conformations

The traditional mesoscopic paradigm represents DNA as a series of base-pair steps whose energy response to equilibrium perturbations is elastic, with harmonic oscillations (defining local stiffness) around a single equilibrium conformation. In addition, base sequence effects are often analysed as a succession of independent XpY base-pair steps (i.e. a nearest-neighbour (NN) model with only 10 unique cases). Unfortunately, recent massive simulations carried out by the ABC consortium suggest that the real picture of DNA flexibility may be much more complex. The paradigm of DNA flexibility therefore needs to be revisited. In this article, we explore in detail one of the most obvious violations of the elastic NN model of flexibility: the bimodal distributions of some helical parameters. We perform here an in-depth statistical analysis of a very large set of MD trajectories and also of experimental structures, which lead to very solid evidence of bimodality. We then suggest ways to improve mesoscopic models to account for this deviation from the elastic regime.     

CD44v6 regulates growth of brain tumor stem cells partially through the AKT-mediated pathway

Identification of stem cell-like brain tumor cells (brain tumor stem-like cells; BTSC) has gained substantial attention by scientists and physicians. However, the mechanism of tumor initiation and proliferation is still poorly understood. CD44 is a cell surface protein linked to tumorigenesis in various cancers. In particular, one of its variant isoforms, CD44v6, is associated with several cancer types. To date its expression and function in BTSC is yet to be identified. Here, we demonstrate the presence and function of the variant form 6 of CD44 (CD44v6) in BTSC of a subset of glioblastoma multiforme (GBM). Patients with CD44(high) GBM exhibited significantly poorer prognoses. Among various variant forms, CD44v6 was the only isoform that was detected in BTSC and its knockdown inhibited in vitro growth of BTSC from CD44(high) GBM but not from CD44(low) GBM. In contrast, this siRNA-mediated growth inhibition was not apparent in the matched GBM sample that does not possess stem-like properties. Stimulation with a CD44v6 ligand, osteopontin (OPN), increased expression of phosphorylated AKT in CD44(high) GBM, but not in CD44(low) GBM. Lastly, in a mouse spontaneous intracranial tumor model, CD44v6 was abundantly expressed by tumor precursors, in contrast to no detectable CD44v6 expression in normal neural precursors. Furthermore, overexpression of mouse CD44v6 or OPN, but not its dominant negative form, resulted in enhanced growth of the mouse tumor stem-like cells in vitro. Collectively, these data indicate that a subset of GBM expresses high CD44 in BTSC, and its growth may depend on CD44v6/AKT pathway.     

Expression of the two isoforms of spinach ribulose 1,5-bisphosphate carboxylase activase and essentiality of the conserved lysine in the consensus nucleotide-binding domain

The two isoforms of ribulose 1,2-bisphosphate carboxylase activase (Rbu-P2 carboxylase) from spinach (Spinacea oleracea L.) were individually purified from Escherichia coli transformed with expression vectors for the appropriate cDNAs. Both isoforms catalyzed activation of Rbu-P2 carboxylase (ribulose 1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) and ATP hydrolysis. The kinetics of the two isoforms with respect to ATP concentration were different, in that the 45-kDa polypeptide exhibited a sigmoidal response while a rectangular response was observed with the 41-kDa isoform. These observations suggest that the additional domain at the C terminus of the 45-kDa isoform modulates the ATP regulation of activity. Lysine 169, at the putative ATP-binding site of the 41-kDa form of Rbu-P2 carboxylase activase, was changed to arginine, isoleucine, and threonine by directed mutagenesis. These mutations abolished Rbu-P2 carboxylase activase and ATPase activities, as well as the capability of the protein to bind ATP. These results confirm that lysine 169 is an essential residue.