Том 43, № 6 (2017)

Hundreds of thousands of short open reading frames (sORFs) that are potentially capable of coding peptides containing up to 100 amino acids are found in the genomes of all living organisms. The known sORF-encoded peptides perform such important functions as regulation of morphogenesis in insects, control of embryogenesis in fish, involvement in the process of formation of nitrogen-fixing nodules in plants, and others. However, the coding potential of most sORFs, as well as the number of such peptides in cells, is almost completely unknown. A systematic approach based on the use of bioinformatics algorithms, as well as transcriptional and proteomic profiling, makes it possible to identify sORFs and analyze their coding potential. In this review, we discuss the issues of transcription and translation of sORFs in cells, the features of their search and identification, as well as the analysis of biological functions.

In modern biotechnological science, there is a need for visualization of objects under study at the levels of cells, organelles, and individual molecules. Prominent among imaging methods are the methods based on the detection of fluorescence from the fluorophores with which objects under study are labeled. Fluorescent proteins (FPs) are very popular as genetically encoded fluorescent labels for lifetime imaging of target structures and processes in living systems. One of the key characteristics of FPs is their photostability, i.e., their resistance to photochemical reactions that quench the fluorescence signal. This review describes the currently known molecular mechanisms underlying photobleaching and the methods used to improve the photostability of fluorescent proteins.

Voltage-gated K⁺ and Na⁺ channels are involved in diverse physiological processes including excitability of heart, muscular and neuronal cells, as well as release of hormones and neurotransmitters. These channels have modular structure and contain five membrane domains: four voltage-sensing domains (VSDs) and one pore domain. VSDs of different channels contain unique ligand-binding sites and are considered as potential pharmacological targets. Modular organization of ion channels points to the possibility of NMR structural studies of isolated VSDs apart from the pore. Here, the feasibility of such studies is considered by the example of VSD of human Kv2.1 channel and VSD-I of human Nav1.4 channel. Cell-free protein expression systems based on the S30 bacterial extract from E. coli, which allow us to produce milligram quantities of VSD samples, including their analogues labeled with stable isotopes, were developed. The choice of membrane- mimicking media that provide long-term stability of the native structure of the membrane protein and high-quality of NMR spectra is a crucial step in NMR studies. Screening of various environments showed that the domains of the Kv2.1 and Nav1.4 channels are unstable in media containing phospholipids: micelles of short-chain lipid DC7PC and lipid-detergent bicelles based on zwitterionic or anionic saturated lipids (DMPC and DMPG). It was demonstrated that the optimal media for NMR studies are the mixtures of zwitterionic and weakly cationic detergents (FOS-12/LDAO). The VSD sample of the Nav1.4 channel in FOS- 12/LDAO environment aggregated irreversibly within a few days despite the high-quality spectra. It is likely that VSDs of human K⁺ and Na⁺ channels are not completely autonomous membrane domains and the contacts with other domains of the channel are required for their stabilization.

The insulin receptor-related receptor (IRR) is the only known metabotropic sensor of extracellular alkaline medium involved in the regulation of the acid–base balance in the body. IRR is expressed in certain cell populations of the kidney, stomach, and pancreas that can come into contact with the extracellular fluids with alkaline pH. To study IRR structure and function, we obtained a stable hybridoma cell-line-producing antibody to the extracellular portion of the receptor. The monoclonal antibody isolated from ascitic fluids showed a positive reaction with the antigen in the ELISA test. The minimum working concentration of antibodies was 12.5 ng/mL. The ability of the antibodies to specifically recognize the purified ectodomain of IRR and the fulllength receptor was confirmed by western blot, immunoprecipitation, and immunocytochemistry.

Novel oligonucleotide derivatives containing N-(methanesulfonyl)-phosphoramidate (mesyl phosphoramidate) group have been described. Solid-phase synthesis of these compounds using an automated DNA synthesizer has been performed for the first time, including the Staudinger reaction between methanesulfonyl azide (mesyl azide) and 3′,5′-dinucleoside 2-cyanoethyl phosphite within an oligonucleotide immobilized on the polymer support, which is a product of phosphoramidite coupling. The mesyl phosphoramidate group is stable to the conditions of oligonucleotide synthesis, in particular, during acidic detritylation and subsequent removal of protecting groups and cleavage of an oligonucleotide from the polymer support by concentrated aqueous ammonia or methylamine at 55°C. It has been shown that the stability of complementary duplexes of oligodeoxynucleotides containing the mesyl phosphoramidate group with a single-stranded DNA is not inferior to the stability of native DNA:DNA duplex. Furthermore, mesyl phosphoramidate oligonucleotides are able to form a complementary duplex with RNA, which is only slightly less stable than the equivalent DNA:RNA duplex. This raises the possibility of their application as potential antisense therapeutic agents.

The ¹²⁵I-labeled B-subunit of the cholera toxin ([125I]CT-B, specific activity of 98 Ci/mmol) was prepared. This subunit was shown to be bound to the membranes which were isolated from epithelial cells of a mucous tunic of the rat thin intestine with high affinity (K_d = 3.7 nM). The binding of the labeled protein was inhibited by the unlabeled α₂-interferon (IFN-α₂), α₁-thymosin, (TM-α₁), and the LKEKK synthetic peptide corresponding to the 16–20 sequence of TM-α₁ and the 131–135 sequence of human IFN-α₂ (Ki 1.0, 1.5, and 2.0 nM, respectively), whereas the KKEKL unlabeled synthetic peptide did not inhibit the binding (K_i > 100 μМ). The LKEKK peptide and CT-B were shown to dose-dependently increase an activity of the soluble guanylate cyclase (sGC) in the concentration range from 10 to 1000 nM. Thus, the binding of TM- α₁, IFN-α₂, and the LKEKK peptide to the CT-B receptor on a surface of the epithelial cells of the mucous tunic of the rat thin intestine resulted in an increase in the intracellular level of cGMP.

A novel series of the DBP(n) fluorescent symmetric dimeric bisbenzimidazoles in which the bisbenzimidazole fragments were attached to an oligomeric linker with the 1,4-piperazine residue in its center were prepared. The DBP(n) molecules were distinguished by the number of methylene groups n (where n = 1, 2, 3, 4) in the linker. The DBP(n) synthesis was based on a condensation of the monomeric bisbenzimidazole (MB) with 1,4-piperazinedialkylcarbonic acids. The ability of the DBP(n) dimeric bisbenzimidazoles to form complexes with the double-stranded DNA was demonstrated by a complex of physicochemical methods, including spectroscopy in the visual UV-area, circular dichroism (CD), and fluorescence. The DBP(1–4) molecules were localized in the DNA minor groove by the CD method with the use of cholesteric liquid-crystalline dispersions (CLCD) of the double-stranded DNA. The DBP(n) dimeric bisbenzimidazoles were easily soluble in water, penetrated through cellular and nuclear membranes, and stained DNA in living cells distinct from the previously synthesized DB(n) series.