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Transketolase catalyzes the transfer of a glycolaldehyde residue from ketose (the donor substrate) to aldose (the acceptor substrate). In the absence of aldose, transketolase catalyzes a one-substrate reaction that involves only ketose. The mechanism of this reaction is unknown. Here, we show that hydroxypyruvate serves as a substrate for the one-substrate reaction and, as well as with the xylulose-5-phosphate, the reaction product is erythrulose rather than glycolaldehyde. The amount of erythrulose released into the medium is equimolar to a double amount of the transformed substrate. This could only be the case if the glycol aldehyde formed by conversion of the first ketose molecule (the product of the first half reaction) remains bound to the enzyme, waiting for condensation with the second molecule of glycol aldehyde. Using mass spectrometry of catalytic intermediates and their subsequent fragmentation, we show here that interaction of the holotransketolase with hydroxypyruvate results in the equiprobable binding of the active glycolaldehyde to the thiazole ring of thiamine diphosphate and to the amino group of its aminopyrimidine ring. We also show that these two loci can accommodate simultaneously two glycolaldehyde molecules. It explains well their condensation without release into the medium, which we have shown earlier.

Conjugates of 17α-substituted testosterone (1 and 2) and 17β-substituted epitestosterone (3 and 4) with pyropheophorbide a were synthesized. The scheme consisted of synthesis of 17α-hydroxy-3-oxopregn-4-en-21-oic and 17β-hydroxy-3-oxopregn-4-en-21-oic acids, and their coupling with pyropheophorbide a by means of either ethylene diamine, or 1,5-diamino pentane linkers. Mutual influence of steroidal and macrocyclic fragments in conjugates molecules was dependent on configuration of C17 and length of linker, that was established by analysis of 1H NMR spectra and molecular models of conjugates. Studies of interaction of conjugates with prostate carcinoma cells revealed that their uptake and internalization were independent on the androgen receptor activity, but dependent on the structure of conjugates, decreasing in the following row: 3 > 4 ≥ 1 > 2. Conjugates significantly decreased the LNCaP and PC-3 cells growth at 96 h incubation. Epitestosterone derivatives 3 and 4 also showed superior anti-proliferative activity versus testosterone ones. Conformationally more rigid conjugates 1 and 3, comprising short linkers, were more active than those with long linkers; conjugate 3 was the most potent.

During recent decades significant progress in studies of the molecular basis of socially significant diseases has been achieved due to introduction of high-throughput methods of genomics and proteomics. Numerous studies, performed within the global program “Human Proteome,” were aimed at identifying all possible proteins in various (including cancer) cell cultures and tissues. One of the aims was to identify socalled biomarkers—the proteins, specific for certain pathologies. However, many studies have shown that the development of the disease is not associated with appearance of new proteins, but it depends on the expression level of certain genes or specific proteoforms representing splice variants, single amino acid polymorphism (SAP) and post-translational modifications (PTM) of proteins. PTMs can play a key role in the development of pathology, because they activate various regulatory or structural proteins in most cellular processes. Among such modifications, phosphorylation appears to be the most significant PTM. This review considers methods of analysis of protein phosphorylation used in studies of the molecular basis of oncological diseases; it contains examples illustrating contribution of modified proteins directly involved in their development as well as examples of screening of such crucial PTMs in diagnostics and selection of methods for treatment.

Study of proteomic composition of exhaled breath condensate (EBC) is a promising non-invasive method for diagnostics of respiratory system diseases in patients. In this study the EBC proteomic composition of 53 donors, including patients with different respiratory system diseases has been investigated. Cytoskeletal keratins type II (1, 2, 3, 4, 5, 6) and cytoskeletal keratins type I (9, 10, 14, 15, 16) were invariant for all samples. Analyzing the frequency of occurrence of proteins in different groups of examined patients, several categories of proteins have been recognized: proteins found in all pathologies (dermcidin, alpha-1- microglobulin, SHROOM3), proteins simultaneously found in two groups (CSTA, LCN1, JUP, PIP, TXN), and proteins specific for a particular group (PRDX1, annexin A1/A2). The EBC analysis by HPLC-MS/MS can be used for identification of potential protein markers specific for inflammatory pulmonary diseases of infective origin (pneumonia) as well as for non-infectious diseases such as chronic obstructive pulmonary disease (COPD).

Phosphorylation is one of the most common and the most important posttranslational modifications (PTM) of proteins. The study of protein phosphoforms is complicated by their low abundance in analyzed biological samples compared with the abundance of corresponding unmodified proteins. The method of selected reactions monitoring (SRM) allows to perform very sensitive and selective PTM analysis with high specificity and sensitivity. Using myelin basic protein (MBP) as a model we have developed a method for phosphoprotein detection by SRM. The method is based on obtaining phosphoproteins in a reconstituted kinase system and following usage of tryptic fragments of the phosphorylated protein as a template for the development of the SRM method. The developed method has been successfully applied for detection of phosphopeptides of myelin basic protein in human brain glioma samples.

This work was aimed at estimating the concentrations of proteins encoded by human chromosome 18 (Chr 18) in plasma samples of 54 healthy male volunteers (aged 20–47). These young persons have been certified by the medical evaluation board as healthy subjects ready for space flight training. Over 260 stable isotope-labeled peptide standards (SIS) were synthesized to perform the measurements of proteins encoded by Chr 18. Selected reaction monitoring (SRM) with SIS allowed an estimate of the levels of 84 of 276 proteins encoded by Chr 18. These proteins were quantified in whole and depleted plasma samples. Concentration of the proteins detected varied from 10–6 M (transthyretin, P02766) to 10–11 M (P4-ATPase, O43861). A minor part of the proteins (mostly representing intracellular proteins) was characterized by extremely high inter individual variations. The results provide a background for studies of a potential biomarker in plasma among proteins encoded by Chr 18. The SRM raw data are available in Proteom...

Direct electrochemistry and bioelectrocatalysis of a newly discovered C-19 steroid 1α-hydroxylase (CYP260A1) from the myxobacterium Sorangium cellulosum So ce56 were investigated. CYP260A1 was immobilized on screen-printed graphite electrodes (SPE) modified with gold nanoparticles, stabilized by didodecyldimethylammonium bromide (SPE/DDAB/Au). Cyclic voltammograms in argon-saturated substrate free 0.1 M potassium phosphate buffer, pH 7.4, and in enzyme-substrate complex with androstenedione demonstrated a redox processes with a single redox couple of E0′ of −299 ± 16 mV and −297.5 ± 21 mV (vs. Ag/AgCl), respectively. CYP260A1 exhibited an electrocatalytic activity detected by an increase of the reduction current in the presence of dissolved oxygen and upon addition of the substrate (androstenedione) in the air-saturated buffer. The catalytic current of the enzyme correlated with substrate concentration in the electrochemical system and this dependence can be described by electrochemical Michaelis-Menten model. The products of CYP260A1-depended electrolysis at controlled working electrode potential of androstenedione were analyzed by mass-spectrometry. MS analysis revealed a mono-hydroxylated product of CYP260A1-dependent electrocatalytic reaction towards androstenedione.

As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration.

The facile synthesis of six [17(20) Z ]- and [17(20) E ]-isomeric 3β-hydroxy-pregna-5,17(20)-dien-21-oyl amides and three [17(20) E ]-3β-hydroxy-2-[prergna-5,17(20)-dien-20-yl]-oxazolines from pregnenolone is presented. The synthetic scheme consists of transformation of pregnenolone into the known 17α-bromo-21-iodo-3β-acetoxypregn-5-en-20-one followed by reaction with ethanolamine, 2-methyl-2-aminopropanol, and (1-aminocyclohexyl)methanol resulted in mixture of [17(20) E ]- and [17(20) Z ]-pregna-5,17(20)-dien-21-(2-hydroxy)-oyl amides; separation of [17(20) E ]- and [17(20) Z ]-isomers; their cyclization into [17(20) E ]-oxazolines under action of POCl 3 in pyridine, and removal of acetate protecting groups. Significantly different orientation of nitrogen containing substituents in [17(20) Z ]- and [17(20) E ]-isomers regarding to steroid backbone enables their configuration to be easily identified by NMR spectroscopy. All synthesized compounds did not exhibit marked toxic effects in three cell lines (MCF-7, Hep G2, and LNCaP). In androgen-sensitive LNCaP cells all testing compounds at concentrations of 50 nM potently stimulated proliferation.

Reaction of 17alpha-bromo-21-iodo-3beta-acetoxypregn-5-en-20-one with ammonia, primary, and secondary amines is simple and convenient method for preparation of [17(20)E]- and [17(20)Z]-pregna-5,17(20)-dien-21-oylamides. Synthesis and characteristics of 12 related amides are presented. Primary testing on cells proliferation indicated differing effects of synthesized compounds on androgen insensitive MCF-7 cells and androgen sensitive LNCaP cells.

The chemical synthesis of six lipophilic conjugates of chlorins was carried out, in which lipophilic fragment (either hexadecyl- or cholest-5-en-3β-yloxyethyl-) bound to 13(1)-, 15(2)-, 17(3)-positions of macrocycle by formation of related carboxamides. Structure of synthesized conjugates was studied by spectral methods and molecular modeling. Lipophilic conjugates of chlorins, being mixed with egg yolk phosphatidyl choline, formed mixed micelles stable in aqueous media under physiological conditions. Mixed micelles of conjugates with phosphatidyl choline differing in stoichiometric compositions were prepared and characterized by absorption spectra, electron microscopy and laser scattering. These micelles were found to bind and internalized by human breast carcinoma MCF-7 cells. The presented data reveal that modification of macrocycle with lipophilic substituents, solubilization of obtained conjugates in aqueous medium as mixed micelles with phospholipids, and transfer of mixed micelles to cells is simple approach for targeting of chlorin derivatives, which apparently may be used in photodynamic therapy.