Anna Zawadzka

Optimization of random time domain sampling in multidimensional NMR.

The detailed description of rules for generation of different random sampling schemes is shown and discussed with regard to Multidimensional Fourier Transform (MFT). The influence of different constrained random sampling schedules on FT of constant signal, i.e., Point Spread Function (PSF), is analyzed considering artifacts level and distribution. We found that Poisson disk sampling schedule, which provides a large low-artifact area in the signal vicinity, is the method of choice in the case of nonlinear sampling of time domain in NMR experiments. We have verified the new sampling schemes by application to the 3D HNCACB and 15N-edited NOESY-HSQC spectra acquired for 13C,15N labeled ubiquitin sample.

Narrow peaks and high dimensionalities: exploiting the advantages of random sampling.

Level of artifacts in spectra obtained by Multidimensional Fourier Transform has been studied, considering randomly sampled signals of high dimensionality and long evolution times. It has been shown theoretically and experimentally, that this level is dependent on the number of time domain samples, but not on its relation to the number of points required in appropriate conventional experiment. Independence of the evolution time domain size (in the terms of both: dimensionality and evolution time reached), suggests that random sampling should be used rather to design new techniques with large time domain than to accelerate standard experiments. 5D HC(CC-TOCSY)CONH has been presented as the example of such approach. The feature of Multidimensional Fourier Transform, namely the possibility of calculating spectral values at arbitrary chosen frequency points, allowed easy examination of resulting spectrum. We present the example of such approach, referred to as Sparse Multidimensional Fourier Transform.

Characterization of a Bacillus subtilis transporter for petrobactin, an anthrax stealth siderophore

Iron deprivation activates the expression of components of the siderophore-mediated iron acquisition systems in Bacillus subtilis, including not only the synthesis and uptake of its siderophore bacillibactin but also expression of multiple ABC transporters for iron scavenging using xenosiderophores. The yclNOPQ operon is shown to encode the complete transporter for petrobactin (PB), a photoreactive 3,4-catecholate siderophore produced by many members of the B. cereus group, including B. anthracis. Isogenic disruption mutants in the yclNOPQ transporter, including permease YclN, ATPase YclP, and a substrate-binding protein YclQ, are unable to use either PB or the photoproduct of FePB (FePBν) for iron delivery and growth, in contrast to the wild-type B. subtilis. Complementation of the mutations with the copies of the respective genes restores this capability. The YclQ receptor binds selectively iron-free and ferric PB, the PB precursor, 3,4-dihydroxybenzoic acid (3,4-DHB), and FePBν with high affinity; the ferric complexes are seen in ESI-MS, implying strong electrostatic interaction between the protein-binding pocket and siderophore. The first structure of a Gram-positive siderophore receptor is presented. The 1.75-Å crystal structure of YclQ reveals a bilobal periplasmic binding protein (PBP) fold consisting of two α/β/α sandwich domains connected by a long α-helix with the binding pocket containing conserved positively charged and aromatic residues and large enough to accommodate FePB. Orthologs of the B. subtilis PB-transporter YclNOPQ in PB-producing Bacilli are likely contributors to the pathogenicity of these species and provide a potential target for antibacterial strategies.

Siderophore-mediated iron acquisition systems in Bacillus cereus: identification of receptors for anthrax virulence-associated petrobactin

During growth under iron limitation, Bacillus cereus and Bacillus anthracis, two human pathogens from the Bacillus cereus group of Gram-positive bacteria, secrete two siderophores, bacillibactin (BB) and petrobactin (PB), for iron acquisition via membrane-associated substrate-binding proteins (SBPs) and other ABC transporter components. Since PB is associated with virulence traits in B. anthracis, the PB-mediated iron uptake system presents a potential target for antimicrobial therapies; its characterization in B. cereus is described here. Separate transporters for BB, PB, and several xenosiderophores are suggested by (55)Fe-siderophore uptake studies. The PB precursor, 3,4-dihydroxybenzoic acid (3,4-DHB), and the photoproduct of FePB (FePB(nu)) also mediate iron delivery into iron-deprived cells. Putative SBPs were recombinantly expressed, and their ligand specificity and binding affinity were assessed using fluorescence spectroscopy. The noncovalent complexes of the SBPs with their respective siderophores were characterized using ESI-MS. The differences between solution phase behavior and gas phase measurements are indicative of noncovalent interactions between the siderophores and the binding sites of their respective SBPs. These studies combined with bioinformatics sequence comparison identify SBPs from five putative transporters specific for BB and enterobactin (FeuA), 3,4-DHB and PB (FatB), PB (FpuA), schizokinen (YfiY), and desferrioxamine and ferrichrome (YxeB). The two PB receptors show different substrate ranges: FatB has the highest affinity for ferric 3,4-DHB, iron-free PB, FePB, and FePB(nu), whereas FpuA is specific to only apo- and ferric PB. The biochemical characterization of these SBPs provides the first identification of the transporter candidates that most likely play a role in the B. cereus group pathogenicity.

Determination of Spin-Spin Couplings from Ultrahigh Resolution 3D NMR Spectra Obtained by Optimized Random Sampling and Multidimensional Fourier Transformation

An example of precise evaluation of backbone scalar J couplings using random sampling of evolution time space in 3D NMR experiments is presented. The recorded spectrum, due to violation of the Nyquist theorem limitation, exhibits ultrahigh resolution in indirect dimensions compared to standard NMR experiment acquired at the same time. The obtained results enable simple and accurate evaluation of scalar and residual dipolar couplings from a single multidimensional NMR experiment.

Highly selective inhibition of butyrylcholinesterase by a novel melatonin–tacrine heterodimers

Novel inhibitors of cholinesterases, especially butyrylcholinesterase (BuChE), were obtained by coupling melatonin–tacrine heterodimers via the carbamate bond. Compounds 14a‐i possessed potent cholinesterase inhibitory activity (with IC50 values as low as 1.18 nm for acetylcholinesterase (AChE) and 0.24 nm for butyrylcholinesterase (BuChE)). These heterodimers exhibit selectivity toward BuChE, being from 4‐ to 256‐fold more active toward BuChE than AChE, but still acting as better AChE inhibitors than tacrine 4.

Immune interference in Mycobacterium tuberculosis intracellular iron acquisition through siderocalin recognition of carboxymycobactins.

The innate immune system antibacterial protein Siderocalin (Scn) binds ferric carboxymycobactin (CMB) and also several catecholate siderophores. Although the recognition of catecholates by Scn has been thoroughly investigated, the binding interactions of Scn with the full spectrum of CMB isoforms have not been studied. Here we show that Scn uses different binding modes for the limited subset of bound CMB isoforms, resulting in a range of binding affinities that are much weaker than other siderophore targets of Scn. Understanding the binding interaction between Scn and CMBs provides clues for the influence of Scn on mycobacterial iron acquisition.

Molecular cloning of melatonin 3-hydroxylase and its production of cyclic 3-hydroxymelatonin in rice (Oryza sativa).

Melatonin is metabolized in animals to cyclic 3‐hydroxymelatonin (3‐OHM) not by an enzymatic pathway, but by interaction with hydroxyl radicals. The production of 3‐OHM in animals suggests the possible presence of 3‐OHM in plants. Prior to the identification of 3‐OHM in plants, we directly cloned the corresponding gene(s) responsible for 3‐OHM synthesis using Escherichia coli library strains expressing genes belonging to the 2‐oxoglutarate‐dependent dioxygenase (2‐ODD) superfamily from rice. Three of 35 E. coli library strains supplemented with 1 mmol/L melatonin were found to produce 3‐OHM in their extracellular medium, suggestive of three 2‐ODD genes involved in 3‐OHM production. The purified recombinant 2‐ODD 11, 2‐ODD 26, and 2‐ODD 33 proteins were shown to catalyze the metabolism of melatonin to 3‐OHM, with 2‐ODD 11 showing the highest melatonin 3‐hydroxylase (M3H) catalytic activity. Consistent with the presence of M3H genes, rice leaves supplemented with 5 mmol/L melatonin produced 3‐OHM [233 μg/g fresh weight (FW)], 2‐hydroxymelatonin (21 μg/g FW), and N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (5 μg/g FW). Three M3H transcripts were induced upon the treatment of rice leaves with cadmium followed by an increase in M3H enzyme activity. Cloning of M3H genes in plants has paved the way for the studies of melatonin in plants in terms of its multiple physiological roles.

The oxidation products of melatonin derivatives exhibit acetylcholinesterase and butyrylcholinesterase inhibitory activity

Abstract:  It is already well documented that melatonin exhibits strong antioxidant properties. It traps several reactive oxygen species including singlet oxygen, peroxyl and hydroxyl radicals. Also, peroxynitrite‐induced reactions are inhibited by melatonin. The oxidation of melatonin by singlet molecular oxygen [O2 (1Δg)] may produce cyclic 3‐hydroxymelatonin whose structure we have already studied. In this investigation we report on the synthesis of several melatonin analogues having a carbamate substituent instead of the methoxy group at 5 position of the indole ring. These compounds behave analogously to melatonin with respect to singlet oxygen and produce the corresponding cyclic 3‐hydroxymelatonin analogues. The structures of the products were investigated with spectral methods and X‐ray crystallography. The compounds obtained possess the 2,3,8,8a‐tetrahydropyrrolo[2,3‐b]indole heterocyclic system which is a structural motif characteristic of alkaloids, physostigmine and phenserine, that are potent acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors used in the Alzheimer’s disease treatment. We measured the inhibitory activity of the obtained compounds against AChE and BChE from human erythrocytes and serum. In the case of the compounds having a phenylcarbamate and methoxyphenylcarbamate substituents, the inhibitory activity (IC50) ranged from 0.252 ± 0.033 to 3.804 ± 0.581 μm. Other compounds were less active and showed rather complex interactions with the structure–activity relationship in need of further investigation.

Simultaneous induction and blockade of autophagy by a single agent

Besides cell death, autophagy and cell senescence are the main outcomes of anticancer treatment. We demonstrate that tacrine-melatonin heterodimer C10, a potent anti-Alzheimer’s disease drug, has an antiproliferative effect on MCF-7 breast cancer cells. The main cell response to a 24 h-treatment with C10 was autophagy enhancement accompanied by inhibition of mTOR and AKT pathways. Significantly increased autophagy markers, such as LC3B- and ATG16L-positive vesicles, confirmed autophagy induction by C10. However, analysis of autophagic flux using mCherry-GFP-LC3B construct revealed inhibition of autophagy by C10 at the late-stage. Moreover, electron microscopy and analysis of colocalization of LC3B and LAMP-1 proteins provided evidence of autophagosome-lysosome fusion with concomitant inhibition of autolysosomal degradation function. After transient treatment with IC50 dose of C10 followed by cell culture without the drug, 20% of MCF-7 cells displayed markers of senescence. On the other hand, permanent cell treatment with C10 resulted in massive cell death on the 5th or 6th day. Recently, an approach whereby autophagy is induced by one compound and simultaneously blocked by the use of another one has been proposed as a novel anticancer strategy. We demonstrate that the same effect may be achieved using a single agent, C10. Our findings offer a new, promising strategy for anticancer treatment.