Том 53, № 4 (2019)

The phenomenon of bacterial persistence, also known as non-inherited antibiotic tolerance in a part of bacterial populations, was described more than 70 years ago. This type of tolerance contributes to the chronization of infectious diseases, including tuberculosis. Currently, the emergence of persistent cells in bacterial populations is associated with the functioning of some stress-induced molecular triggers, including toxin–antitoxin systems. In the presented review, genetic and metabolic peculiarities of persistent cells are considered and the mechanisms of their occurrence are discussed. The hypothesis of the origin of persister cells based on bistability, arising due to the non-linear properties of a coupled transcription–translation system, was proposed. Within this hypothesis, the phenomenon of the bacterial persistence of modern cells is considered as a result of the genetic fixation of the phenotypic multiplicity that emerged in primitive cells in the process of neutrally coupled co-evolution (genetic drift of multiple neutrally coupled mutations). Our hypothesis explains the properties of persister cells, as well as their origin and “ineradicable” nature.

The protein synthesis in cells occurs in ribosomes, with the involvement of protein translational factors. One of these translational factors is the elongation factor P (EF-P). EF-P is a three-domain protein that binds between the P and E sites of the ribosome, near the P-tRNA, the peptidyl transferase center, and E-site codon of the mRNA. The majority of studies showed that the EF-P helps the ribosome to synthesize stalling amino acid motifs, such as polyprolines. In the first part of this review, we inspect the general evolutionary variety of the EF-P in different organisms, the problems of the regulation provided by the EF-P, and its role in the sustainability of the protein balance in the cell in different physiological states. Although the functions of the EF-P have been well studied, there are still some problems that remain to be solved. The data from recent studies contradict the previous theories. Consequently, in the second part, we discuss the recent data that suggest the involvement of the EF-P in each translocation event, not only in those related to polyproline synthesis. This activity contradicts some aspects of the known pathway of the removal of the E-tRNA during the translocation event. In addition, in the third part of this review, we tried to partly shift the interest from the antistalling activity of domain I of the EF-P to the action of domain III, the functions of which has not been closely studied. We expand on the idea about the involvement of domain III of the EF-P in preventing the frameshift and debate the EF-P’s evolutionary history.

A new plasmid, pSM22, was isolated from Serratia marcescens and sequenced. Its 43 190-bp sequence with an average GC-content of 58% contains 31 open reading frames (ORFs) which form replication, conjugation, stability, and adaptive modules. The replication module includes a site of initiation of leading-strand synthesis in plasmid replication, a replication termination site (terC), the repA (=repA1) and repA4 genes, and the copA sequence, which codes for an antisense RNA (asRNA). These structures are functionally integrated in an FII replicon (incompatibility group IncFII). Based on the significant differences between the FII replicon and the canonical sequences of the R plasmids R1 and NR1 (=R100=R222), pSM22 was assigned to a new subtype. The conjugation module includes 13 genes with a high identity to the genes responsible for conjugation of the F plasmid. A comparative genomic analysis showed that the conjugation modules of pSM22 and F are structurally similar. By the conjugation system and the presence of three conserved motifs in relaxase (TraI), pSM22 belongs to the F12 clade of the MOB_F type. The stability module includes the resD and parA genes, which are responsible for the resolution of multimeric plasmid forms and their subsequent segregation between daughter cells. The adaptive module contains the microcin H47 (MccH47) secretion/processing and UV resistance genes. The mosaic structure of pSM22 and reductive evolution of its modules suggest high genomic plasticity for the genus Serratia. An analysis of the architecture of the pSM22 modules clarifies the evolutionary relationships among IncF/MOB_F12 group plasmids in bacteria of the family Enterobacteriaceae and opens a novel avenue for further comparative genomic studies of Serratia plasmids.

The woolly mammoth mitochondrial genome (including the Malolyakhovsky mammoth) has been previously sequenced, followed by the annotation of all its genes (MF770243). In this study, based on the Malolyakhovsky mammoth, we describe for the first time the sites of functional significance in the control region of the woolly mammoth mitogenome.

The ubiquitin-proteasome system (UPS) performs proteolysis of most intracellular proteins. The key components of the UPS are the proteasomes, multi-subunit protein complexes, playing an important role in cellular adaptation to various types of stress. We analyzed the dynamics of the proteasome activity, the content of proteasome subunits, and the expression levels of genes encoding catalytic subunits of proteasomes in the human histiocytic lymphoma U937 cell line immediately, 2, 4, 6, 9, 24, and 48 h after a heat shock (HS). The initial decrease (up to 62%) in the proteasome activity in cellular lysates was revealed, then 10 h after HS the activity began to recover. The amount of proteasomal α-subunits in the cells decreased 2 h after HS, and was restored to 24–48 h after HS. Fluctuations in the levels of mRNAs encoding proteasome catalytic subunits with the maximum expression 2 h after HS and a gradual decrease to 48 h after HS were observed. The average estimated number of mRNA copies per cell ranged from 10 for weakly to 150 for highly expressed proteasome genes. Thus, the recovery efficiency of UPS functionality after HS, which reflects the important role of proteasomes in maintaining cell homeostasis, was evaluated.

Malignant cutaneous melanoma (CM) is an extremely aggressive cancer characterized by a high level of metastatic activity and unfavorable prognosis due to a high incidence of relapses, as well as resistance to standard chemotherapy. Cutaneous melanoma accounts for 80% of deaths from malignant skin tumors. Nucleolin/C23 and nucleophosmin/B23, which constitute altogether ~70% of the nucleolus volume, are promising targets for molecular therapy of melanoma. These proteins perform many important functions in the cell, so disruption of the NCL and/or NPM gene structure and abnormal expression of the C23 and B23 proteins they encode, can lead to unlimited cell proliferation and progression of a tumor. Therefore, investigation of the structure and expression of these genes is a topical problem, which is important for understanding the mechanisms of CM carcinogenesis and for the development of new therapeutic approaches. This paper describes new NCL and NPM polymorphisms, as well as the levels of C23 and B23 expression in normal tissues, CM and mucosal melanoma.

Acriflavine resistance protein B (AcrB) serves as prototype for multidrug resistance (MDR) efflux transporters of resistance nodulation division (RND) superfamily. AcrB has been proven as potential drug target with many synthetic and natural inhibitors have been identified such as those belonging to pyranopyridine, naphthamide and pimozide classes. The plant derived alkaloid inhibitors represented by reserpine has been found to inhibit both ATP binding cassette and major facilitator efflux transporters. In this study we report the reserpine induced inhibition of RND transporter AcrB. The preliminary docking analysis hints that reserpine shares its binding site with ciprofloxacin, a known substrate of AcrB and could possibly act as competitive inhibitor. For in vitro validation, AcrB from Salmonellatyphi was cloned under the control of tac promoter and resulting vector was introduced into E. coli C41(DE3). Under autoinduced conditions, cells overexpressing AcrB transporter were subjected to combined dose of ciprofloxacin and reserpine. The combined exposure resulted in enhanced ciprofloxacin-induced growth inhibition of cells expressing AcrB transporter as compared to control cells transformed with vector of backbone sequence. Time kill analysis further confirmed these findings. To the best of our knowledge, this is first study to show that exposure to reserpine induces inhibition of AcrB. The assay developed in this study allows simple and reproducible detection of substrate/inhibitor effects upon AcrB and related efflux transporters.

In the past decade, mass spectrometry studies of skeletal muscles have become common. In this tissue, the abundance of several contractile proteins significantly limits the depth of the panoramic proteome analysis. The use of isobaric labels allows improving assessment of the changes in the protein content, while analyzing up to 10 samples in a single run. Here we present the results of a comparative study of various methods for the fractionation of skeletal muscle peptides labeled with an isobaric label iTRAQ. Samples from m. vastus lateralis of eight young males were collected with a needle biopsy. After digestion into peptides and labeling, the preparations were carried out according to three different protocols: (1) peptide purification, HPLC-MS/MS; (2) peptide purification, isoelectric focusing, HPLC-MS/MS; (3) high pH reverse-phase LC fractionation, HPLC-MS/MS. Fractionation of labeled peptides by high pH reverse-phase LC was the optimal strategy for increasing the depth of the proteome analysis. This approach, in addition to contractile and mitochondrial proteins, allowed us to detect a variety of regulatory molecules, including the nucleic acids binding the proteins, chaperones, receptors, and transcription factors.

miRNAs regulate the expression of many genes and are involved in the development of diseases. We studied miRNAs that interact partly or fully complementarily with the 5'UTR, CDS and 3'UTR of mRNAs of target genes. The MirTarget program used in this study allows for the discovery of miRNA binding sites (BS) in the entire nucleotide sequence of the mRNA and for determining the characteristics of the interactions of miRNAs with mRNAs. We identified five pairs of fully complementary BS for miR-127-5p and miR-127-3p, miR-136-5p and miR-136-3p, miR-431-5p and miR-431-3p, miR-432-5p and miR-432-3p, and miR-433-5p and miR-433-3p in the CDS of the human and animal mRNA of RTL1 gene. The fully complementary BS for miR-6720-5p, miR-6720-3p were identified in the CDS of the FOXF2 gene; BS for miR-3187-5p, miR-3187-3p were found in the CDS of the PLPPR3 gene; BS for miR-4665-5p, miR-4665-3p were found in the 5′UTR of the KIAA2026 gene; BS for miR-135a-5p, miR-135a-3p were found in the 3′UTR of the GLYCTK gene; BS for miR-7106-5p, miR-7106-3p were found in the 3′UTR of the CCDC42B gene. The miRNA-5p and miRNA-3p associated with the RTL1 gene have BS in the mRNAs of 32 target human genes. The miRNA-5p and miRNA-3p associated with the FOXF2, PLPPR3, KIAA2026, GLYCTK and CCDC42B genes have BS in the mRNAs of 27 target genes, involved in development of several diseases. Nucleotide sequences of miRNA-5p and miRNA-3p and BS are conserved over tens of millions of years of divergence of the studied animal species. Binding characteristics of miR-3120-3p and miR-3120-5p, miR-196b-3p and miR-196b-5p, miR-125a-3p and miR-125a-3p, let-7e-3p and let-7e-5p, miR-99b-3p in fully complementary BS of non-coding DMN3OS, HOXA10-AS, SPACA6P-AS genes have been established.