A Structural Biology Approach to Understand Human Lymphatic Filarial Infection

The presence of aspartic protease inhibitor in filarial parasite Brugia malayi (Bm-Aspin) makes it interesting to study because of the fact that the filarial parasite never encounters the host digestive system. Here, the aspartic protease inhibition kinetics of Bm-Aspin and its NMR structural characteristics have been investigated. The overall aim of this study is to explain the inhibition and binding properties of Bm-Aspin from its structural point of view. UV-spectroscopy and multi-dimensional NMR are the experiments that have been performed to understand the kinetic and structural properties of Bm-Aspin respectively. The human aspartic proteases that are considered for this study are pepsin, renin, cathepsin-E and cathepsin-D. The results of this analysis performed with the specific substrate [Phe-Ala-Ala-Phe (4-NO₂)-Phe-Val-Leu (4-pyridylmethyl) ester] against aspartic proteases suggest that Bm-Aspin inhibits the activities of all four human aspartic proteases. The kinetics studies indicate that Bm-Aspin follows a competitive mode of inhibition for pepsin and cathepsin-E, non-competitive for renin and mixed mode for cathepsin-D. The triple resonance NMR experiments on Bm-Aspin suggested the feasibility of carrying out NMR studies to obtain its solution structure. The NMR titration studies on the interactions of Bm-Aspin with the proteases indicate that it undergoes fast-exchange phenomena among themselves. In addition to this, the chemical shift perturbations for some of the residues of Bm-Aspin observed from ¹⁵N-HSQC spectra upon the addition of saturated amounts of aspartic proteases suggest the binding between Bm-Aspin and human aspartic proteases. They also provide information on the variations in the intensities and mode of binding between the proteases duly corroborating with the results from the protease inhibition assay method.     

A molybdopterin-free form of xanthine oxidase

A previously unidentified fraction lacking xanthine:O2 activity has been isolated during affinity chromatography of bovine milk xanthine oxidase preparations on Sepharose 4B/folate gel. Unlike active, desulfo, or demolybdo forms of xanthine oxidase, this form, which typically comprises about 5% of an unfractionated enzyme solution, passes through the affinity column without binding to it, and is thus easily separated from the other species. The absorption spectrum of this fraction is very similar to that of the active form, but has a 7% lower extinction at 450 nm. Analysis of the fraction has shown that it is a dimer of normal size, but that it does not contain molybdenum or molybdopterin (MPT). The "MPT-free" xanthine oxidase contains 90-96% of the Fe found in active xanthine oxidase, and 100% of the expected sulfide. EPR and absorption difference spectroscopy indicate that the MPT-free fraction is missing approximately half of its Fe/S I centers. The presence of a new EPR signal suggests that an altered Fe/S center may account for the nearly normal Fe and sulfide content. Microwave power saturation parameters for the Fe/S II and Fe/S I centers in the MPT-free fraction are normal, with P1/2 equal to 1000 and 60 mW, respectively. The new EPR signal shows intermediate saturation behavior with a P1/2 = 200 mW. The circular dichroism spectrum of the MPT-free fraction shows distinct differences from that of active enzyme. The NADH:methylene blue activity of the MPT-free fraction is the same as that of active xanthine oxidase which exhibits xanthine:O2 activity, but NADH:cytochrome c and NADH:DCIP activities are diminished by 54 and 37%, respectively.     

CB-RAF600E-1 exerts efficacy in vemurafenib-resistant and non-resistant-melanoma cells via dual inhibition of RAS/RAF/MEK/ERK and PI3K/Akt signaling pathways

Background and AimPredicting novel dual inhibitors to combat adverse effects such as the development of resistance to vemurafenib in melanoma treatment due to the reactivation of MAPK and PI3K/AKT signaling pathways is studied to help in reversal of cancer symptoms.Reversal of cancer symptoms in melanoma associated with vemurafenib resistance is driven by reactivation of MAPK and PI3K/Akt signaling pathways. Novel dual inhibitors targeting these proteins would be beneficial to combat resistance.MethodsHigh-throughput virtual screening of the ChemBridge library against B-RAFV600E and Akt was performed using an automated protocol with the AutoDock VINA program. Luminescence and time-resolved fluorescence kits were used to measure enzyme activities. The MTT assay was used to determine proliferation in normal and vemurafenib-resistant A375 cells. Flow cytometry was used to examine apoptosis, cell cycle, and phosphorylation of ERK/Akt signaling pathway.ResultsHigh-throughput screening from the ChemBridge library identified 15 compounds with high binding energy towards B-RAFV600E; among these, CB-RAF600E-1 had the highest ΔG_binding score −11.9 kcal/mol. The compound also had a high affinity towards Akt, with a ΔG_binding score of −11.5 kcal/mol. CB-RAF600E-1 dose-dependently inhibited both B-RAFV600E and Akt with IC₅₀ values of 635 nM and 154.3 nM, respectively. The compound effectively controlled the proliferations of normal and vemurafenib-resistant A375 cells, with GI₅₀ values of 222.3 nM and 230.5 nM, respectively. A dose-dependent increase in the sub G₀/G₁ phase of the cell cycle and total apoptosis was observed following compound treatment in both normal and vemurafenib-resistant melanoma cells. Treatment with CB-RAF600E-1 decreased the pERK/pAkt dual-positive populations in normal and vemurafenib-resistant A375 cells.ConclusionCB-RAF600E-1, identified as a novel dual inhibitor effective against normal and vemurafenib-resistant melanoma cells, requires further attention for development as an effective chemotherapeutic agent for melanoma management.     

Characterization of molybdenum cofactor from Escherichia coli.

Molybdenum cofactor activity was found in the soluble fraction of cell-free extracts of Escherichia coli grown aerobically in media supplemented with molybdate. Cofactor was detected by its ability to complement the nitrate reductase-deficient mutant of Neurospora crossa, nit-1, resulting in the vitro formation of nitrate reductase activity. Acid treatment of E. coli extracts was not required for release of cofactor activity. Cofactor was able to diffuse through a membrane of nominal 2,000-molecular-weight cutoff and was insensitive to trypsin. The cofactor was associated with a carrier molecule (approximately 40,000 daltons) during gel filtration and sucrose gradient centrifugation, but was easily removed from the carrier by dialysis. The carrier molecule protected the cofactor from inactivation by heat or oxygen. E. coli grown in molybdenum-free media, without and with tungsten, synthesized a metal-free "empty" cofactor and its tungsten analog, respectively, both of which were subsequently activated by the addition of molybdate. Empty and tungsten-containing cofactor complemented the nitrate reductase subunits in the nit-1 extract, forming inactive, but intact, 7.9S nitrate reductase. Addition of molybdate to the enzyme complemented in this manner restored nitrate reductase activity.     

Troxerutin suppresses lipid abnormalities in the heart of high-fat–high-fructose diet-fed mice

The reversal effect of troxerutin (TX) on obesity, insulin resistance, lipid accumulation, oxidative damage, and hypertension induced in the high-fat–high-fructose diet (HFFD)-fed mice model of metabolic syndrome was investigated. Adult male Mus musculus mice of body weight 25–30 g were fed either control diet or HFFD. Each group was divided into two and treated or untreated with TX (150 mg/kg bw, p.o.) from the 16th day. Assays were done in plasma and heart after 30 and 60 days of the experimental period. Significant increase in the levels of glucose and insulin, blood pressure (BP), and oxidative stress were observed after 30 days of HFFD feeding as compared to control. Animals fed HFFD for 60 days developed more severe changes in the above parameters compared to those fed for 30 days. Hearts of HFFD-fed mice registered downregulation of peroxisome proliferator-activated receptor-α and peroxisome proliferator-activated receptor gamma coactivator-1α, carnitine palmitoyl transferse-1b and AMP-activated protein kinase; and upregulation of cluster of differentiation 36, fatty acid-binding protein-1, and sterol regulatory element-binding protein-1c after 60 days. TX administration restricted obesity (as seen by Lee’s index); improved whole body insulin sensitivity; reduced BP, lipid accumulation, and oxidative damage; upregulated fatty acid (FA) oxidation; and downregulated FA transport and lipogenesis. Histology of heart revealed that TX diminishes inflammatory cell infiltration and fatty degeneration in HFFD-fed mice. The antioxidant property of TX and its ability to influence lipid regulatory genes could be the underlying mechanisms for its beneficial effects.     

The presence and distribution of reduced folates in Escherichia coli dihydrofolate reductase mutants

Escherichia coli DNA photolyase was overproduced and purified from each of two mutant E. coli strains lacking dihydrofolate reductase. The extent of over-production in the mutants was comparable to that seen in the wild type strain. Examination of the isolated photolyase from these strains revealed that the folate cofactor, 5,10-methenyltetrahydrofolate, was present in these proteins at a level of 60-80% compared to that purified from the wild type strain. Further examination of the dihydrofolate reductase-deficient strains revealed the presence of other tetrahydrofolate derivatives. These findings demonstrate that dihydrofolate reductase is not essential for the production of tetrahydrofolates in E. coli.     

Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins.

An interesting property of the Escherichia coli DNA polymerase II is the stimulation in DNA synthesis mediated by the DNA polymerase III accessory proteins beta,gamma complex. In this paper we have studied the basis for the stimulation in pol II activity and have concluded that these accessory proteins stimulate pol II activity by increasing the processivity of the enzyme between 150- and 600-fold. As is the case with pol III, processive synthesis by pol II requires both beta,gamma complex and SSB protein. Whereas the intrinsic velocity of synthesis by pol II is 20-30 nucleotides per s with or without the accessory proteins, the processivity of pol II is increased from approximately five nucleotides to greater than 1600 nucleotides incorporated per template binding event. The effect of the accessory proteins on the rate of replication is far greater on pol III than on pol II; pol III holoenzyme is able to complete replication of circular single-stranded M13 DNA in less than 20 s, whereas pol II in the presence of the gamma complex and beta requires approximately 5 min. We have investigated the effect of beta,gamma complex proteins on bypass of a site-specific abasic lesion by E. coli DNA polymerases I, II, and III. All three polymerases are extremely inefficient at bypass of the abasic lesion. We find limited bypass by pol I with no change upon addition of accessory proteins. pol II also shows limited bypass of the abasic site, dependent on the presence of beta,gamma complex and SSB. pol III shows no significant bypass of the abasic site with or without beta,gamma complex.