Comprehensive analysis of the molecular docking of small molecule inhibitors to the Aβ1–40 peptide and its Osaka-mutant: insights into the molecular mechanisms of Aβ-peptide inhibition

Abstract

Alzheimer’s disease (AD) is the most common form of age-related neurodegeneration occurs because of deposition of proteins in the form of extracellular plaques containing aggregated amyloid beta (Aβ) peptide and intracellular neurofibrillary tangles composed of aggregated microtubule-binding protein tau. Amyloid aggregation process can be enhanced by several familial AD-associated mutations in Aβ peptide. In this study, we have unravelled the interactions of 40 small molecule inhibitors with the Osaka-mutant of Aβ_1–40 peptide at atomic level and characterized modes of their binding to mutant Aβ by docking approaches. We have also compared docking energies of these inhibitors with Osaka-mutant with those previously determined for the wild-type and Iowa-mutant peptides and discussed in light of the peptide conformations and non-covalent interactions. We have also discussed inhibition mechanisms of these three peptides. Our analyses revealed that these small molecules can efficiently inhibit Osaka-mutant. The binding modes of drugs with these three peptides are markedly different and so are the mechanisms of inhibition of these three peptides. Overall analysis of the data reveals that binding energy of Iowa-mutant drug complex is lowest and most stable which is followed wild-type peptide-drug complex followed by Osaka-mutant drug complex.

Communicated by Ramaswamy H. Sarma     

Endocytosis of Mycobacterium tuberculosis Heat Shock Protein 60 Is Required to Induce Interleukin-10 Production in Macrophages

Understanding the signaling pathways involved in the regulation of anti-inflammatory and pro-inflammatory responses in tuberculosis is extremely important in tailoring a macrophage innate response to promote anti-tuberculosis immunity in the host. Although the role of toll-like receptors (TLRs) in the regulation of anti-inflammatory and pro-inflammatory responses is known, the detailed molecular mechanisms by which the Mycobacterium tuberculosis bacteria modulate these innate responses are not clearly understood. In this study, we demonstrate that M. tuberculosis heat shock protein 60 (Mtbhsp60, Cpn60.1, and Rv3417c) interacts with both TLR2 and TLR4 receptors, but its interaction with TLR2 leads to clathrin-dependent endocytosis resulting in an increased production of interleukin (IL)-10 and activated p38 MAPK. Blockage of TLR2-mediated endocytosis inhibited IL-10 production but induced production of tumor necrosis factor (TNF)-α and activated ERK1/2. In contrast, upon interaction with TLR4, Mtbhsp60 remained predominantly localized on the cell surface due to poorer endocytosis of the protein that led to decreased IL-10 production and p38 MAPK activation. The Escherichia coli homologue of hsp60 was found to be retained mainly on the macrophage surface upon interaction with either TLR2 or TLR4 that triggered predominantly a pro-inflammatory-type immune response. Our data suggest that cellular localization of Mtbhsp60 upon interaction with TLRs dictates the type of polarization in the innate immune responses in macrophages. This information is likely to help us in tailoring the host protective immune responses against M. tuberculosis.     

Characterization of a lysyl aminopeptidase activity associated with phosphoglucose isomerase of Vibrio vulnificus

Phosphoglucose isomerase (PGI) is a multifunctional enzyme involved in glycolysis and gluconeogenesis and, in mammalian cells, functions as neuroleukin, autocrine motility factor (AMF), and differentiation and maturation factor (MF). We isolated and characterized PGI with a novel lysyl aminopeptidase (LysAP) activity (PGI-LysAP) from Vibrio vulnificus. Mass spectrometry revealed that PGI-LysAP is a heterodimer consisting of 23.4- and 60.8-kDa subunits. Only the heterodimer displayed LysAP activity. PGI-LysAP has a pI around 6.0 and high specificity toward the synthetic, fluorogenic substrate l-lysyl-7-amino-4-methylcoumarin. LysAP activity is optimal at pH 8.0, is 64% higher at 37 degrees C than at 21 degrees C, does not directly correlate with virulence, and is strongly inhibited by serine protease and metalloprotease inhibitors. PGI-LysAP was also identified in Vibrio parahaemolyticus and V. cholerae, but was absent from non-Vibrio human pathogens. Sequencing of the pgi gene revealed 1653 bp coding for a 550-amino-acid protein. Cloned and expressed PGI formed a homodimer with isomerase activity, but not LysAP activity. The finding of LysAP activity associated with heterodimeric PGI should foster a broad search for putative substrates in an effort to elucidate the role of PGI-LysAP in bacteria and its roles in the pathophysiology of diseases.     

Rate-coding of spinal motoneurons with high-frequency magnetic stimulation of human motor cortex

Rate-coding in spinal motoneurons was studied using high-frequency magnetic stimulation of the human motor cortex. The subject made a weak contraction to cause rhythmic (i.e., tonic) discharge of a single motor unit in flexor (or extensor) carpi radialis or tibialis anterior, while the motor cortical representation of that muscle was stimulated with brief trains of pulses from a Pyramid stimulator (4 Magstim units connected by 3 BiStim modules). An "m@n" stimulus train consisted of m number of pulses (1-4), with an interpulse interval (IPI) of n ms (1-6). Peristimulus time histograms were constructed for each stimulus condition of a given motor unit, and related to the average rectified surface electromyography (EMG) from that muscle. Surface EMG responses showed markedly more facilitation than single-pulse stimulation, with increasing numbers of pulses in the train; responses also tended to increase in magnitude for the longer IPI values (4 and 6 ms) tested. Motor-unit response probability increased in a manner comparable to that of surface EMG. In particular, motoneurons frequently responded twice to a given stimulus train. In addition to recruitment of new motor units, the increased surface EMG responses were, in part, a direct consequence of short-term rate-coding within the tonically discharging motoneuron. Our results suggest that human corticomotoneurons are capable of reliably following high-frequency magnetic stimulation rates, and that this activity pattern is carried over to the spinal motoneuron, enabling it to discharge at extremely high rates for brief periods of time, a pattern known to be optimal for force generation at the onset of a muscle contraction.     

“Just a spoonful of sugar...”: import of sialic acid across bacterial cell membranes

Eukaryotic cell surfaces are decorated with a complex array of glycoconjugates that are usually capped with sialic acids, a large family of over 50 structurally distinct nine-carbon amino sugars, the most common member of which is N-acetylneuraminic acid. Once made available through the action of neuraminidases, bacterial pathogens and commensals utilise host-derived sialic acid by degrading it for energy or repurposing the sialic acid onto their own cell surface to camouflage the bacterium from the immune system. A functional sialic acid transporter has been shown to be essential for the uptake of sialic acid in a range of human bacterial pathogens and important for host colonisation and persistence. Here, we review the state-of-play in the field with respect to the molecular mechanisms by which these bio-nanomachines transport sialic acids across bacterial cell membranes.     

Adaptation of the Lyme disease spirochaete to the mammalian host environment results in enhanced glycosaminoglycan and host cell binding

The Lyme disease spirochaete, Borrelia burgdorferi, is transmitted to mammals by Ixodes ticks and can infect multiple tissues. Host cell attachment may be critical for tissue colonization, and B. burgdorferi cultivated in vitro recognizes heparin- and dermatan sulphate-related glycosaminoglycans (GAGs) on the surface of mammalian cells. To determine whether growth of the spirochaete in the mammalian host alters GAG binding, we assessed the cell attachment activities of B. burgdorferi grown in vitro or in dialysis membrane chambers implanted intraperitoneally in rats. Host-adapted B. burgdorferi exhibited approximately threefold better binding to purified heparin and dermatan sulphate and to GAGs expressed on the surface of cultured endothelial cells. Three B. burgdorferi surface proteins, Bgp, DbpA and DbpB, have been demonstrated previously to bind to GAGs or to GAG-containing molecules, and we show here that recombinant derivatives of each of these proteins were able to bind to purified heparin and dermatan sulphate. Immunofluorescent staining of in vitro-cultivated or host-adapted spirochaetes revealed that DbpA and DbpB were present on the bacterial surface at higher levels after host adaptation. Recombinant Bgp, DbpA and DbpB each partially inhibited attachment of host-adapted B. burgdorferi to cultured mammalian cells, consistent with the hypothesis that these proteins may promote attachment of B. burgdorferi during growth in the mammalian host. Nevertheless, the partial nature of this inhibition suggests that multiple pathways promote mammalian cell attachment by B. burgdorferi in vivo. Given the observed increase in cell attachment activity upon growth in the mammalian host, analysis of host-adapted bacteria will facilitate identification of the cell binding pathways used in vivo.     

Drug resistance in Indian isolates of enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis>

Five different strains of enteropathogenic Escherichia coli (EPEC) and one non-pathogenic strain of E. coli were studied for the determination of resistance to various antibiotics by disc inhibition test. Our results demonstrated multiple drug resistance among the selected E. coli isolates with all of them including the non-pathogenic control strain showing high resistance towards ampicillin. Our results from conjugation test clearly showed the presence of most of the antibiotic markers on the transferred plasmids. Simultaneously, the plasmid profile of conjugants also indicated the presence of more than one plasmid along with the expected megaplasmid of ~90 Kb present in them. Thus our results amply demonstrate that these drug markers are associated with these mobile plasmids.     

Glutamate Decarboxylase and GABA Aminotransferase Levels in Different Regions of Rat Brain on the Onset of Leptazol Induced Convulsions

The activities of Glutamate decarboxylase (GAD) and Gamma aminobutyric acid (GABA) were studied in three regions of rat brain in heightened neuronal activity resulting in convulsions by Leptazol. These enzymes were studied in preconvulsive, convulsive and post convulsive phases. The activity of GAD decreases significantly in the preconvulsive phase in all the three regions of brain followed by a significant increase during the convulsive and post convulsive phase in cerebral cortex and cerebellum. The activity of GABA-T decreases maximal during the preconvulsive phase followed by convulsive phase. The activity of this enzyme tended to increase to control values when the postconvulsive phase was reached. Therefore, it is suggested that the concomitant decrease of GAD activity and GABA concentration, is probably an important factor in the onset of convulsions.     

Spleen necrosis virus-derived C-type retroviral vectors for gene transfer to quiescent cells

Gene therapy applications of retroviral vectors derived from C-type retroviruses have been limited to introducing genes into dividing target cells. Here, we report genetically engineered C-type retroviral vectors derived from spleen necrosis virus (SNV), which are capable of infecting nondividing cells. This has been achieved by introducing a nuclear localization signal (NLS) sequence into the matrix protein (MA) of SNV by site-directed mutagenesis. This increased the efficiency of infecting nondividing cells and was sufficient to endow the virus with the capability to efficiently infect growth-arrested human T lymphocytes and quiescent primary monocyte-derived macrophages. We demonstrate that this vector actively penetrates the nucleus of a target cell, and has potential use as a gene therapy vector to transfer genes into nondividing cells.     

Photosynthetic Parameters at the Vegetative Stage and during Grain Development of Two Hexaploid Wheat Cultivars Differing in Salt Tolerance

Response of two spring wheat (Triticum aestivum L.) cultivars, salt tolerant SARC-I and salt sensitive Potohar, to different concentrations of NaCl was examined under glasshouse conditions. Eighteen-day-old plants of both the lines grown in sand culture were irrigated with 0 (control), 80, 160 or 240 mM NaCl in full strength Hoagland's nutrient solution. Shoot fresh and dry masses, and leaf area per plant of SARC-I at the vegetative stage, were significantly greater than those of cv. Potohar at higher salt concentrations, however, relative growth rate (RGR) of cv. Potohar was significantly higher than that of SARC-I. SARC-I had higher net photosynthetic rate (P_N), stomatal conductance (g_s) and transpiration rate (E) than cv. Potohar at the vegetative stage, but the cultivars did not differ significantly in water-use efficiency (P_N/E), intrinsic water use efficiency (P_N/g_s), and intercellular/ambient CO₂ concentration ratio. At the grain development stage, SARC-I had significantly higher P_N and g_s in the flag leaf than cv. Potohar under salinity. SARC-I was superior to cv. Potohar with respect to number of grains per spike, number of grains per spikelet, mean grain mass, and grain yield per plant at all NaCl concentrations.