Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.11, #1)

Peptide phage display in biotechnology and biomedicine by G. A. Kuzmicheva; V. A. Belyavskaya (1-15).
To date peptide phage display is one of the most common combinatorial methods used for identifying specific peptide ligands. Phage display peptide libraries containing billions different clones successfully used for selection of ligands with high affinity and selectivity toward wide range of targets including individual proteins, bacteria, viruses, spores, different kind of cancer cells and variety of nonorganic targets (metals, alloys, semiconductors, etc.). Success of using filamentous phage in phage display technologies relays on the robustness of phage particles and a possibility to genetically modify its DNA to construct new phage variants with novel properties. In this review we are discussing characteristics of the most known non-commercial peptide phage display libraries of different formats (landscape libraries in particular) and their successful applications in several fields of biotechnology and biomedicine: discovery of peptides with diagnostic values against different pathogens, discovery and using of peptides recognizing cancer cells, trends in using of phage display technologies in human interactome studies, application of phage display technologies in construction of novel nanomaterials.
Keywords: phage display; peptides; bacteriophages; random peptide phage libraries; landscape libraries

The role of atypical ubiquitination in cell regulation by O. A. Buneeva; A. E. Medvedev (16-31).
Ubiquitination is a type of intracellular proteins post-translational modification (PTM) characterized by covalent attachment of ubiquitin molecules to target proteins. This includes monoubiquitination (attachment of one ubiquitin molecule), multiple monoubiquitination also known as multiubiquitination (attachment of several monomeric ubiquitin molecules to a target protein), and polyubiquitination (attachment of ubiquitin chains consisting of several, most frequently four ubiquitin monomers to a target protein). In the case of polyubiquitination, linear or branched polyubiquitin chains are formed. Their formation involves various lysine residues of monomeric ubiquitin. The best studied is Lys48-linked polyubiquitination, which targets proteins for proteasomal degradation. In this review we have considered examples of so-called atypical polyubiquitination, which mainly involves other lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys63) and also N-terminal methionine. The considered examples convincingly demonstrate that polyubiquitination of proteins (not necessarily) targets proteins for their proteolytic degradation in proteasomes. Atypically polyubiquitinated proteins are involved in regulation of various processes including immune response, genome stability, signal transduction, etc. Alterations of ubiquitination machinery is crucial for development of serious diseases.
Keywords: ubiquitin; posttranslational modification of proteins; atypical ubiquitination; ubiquitination enzymes

ADAR-mediated messenger RNA Editing: Analysis at the proteome level by A. A. Kliuchnikova; K. G. Kuznetsova; S. A. Moshkovskii (32-42).
Post-transcriptional RNA editing by RNA-specific adenosine deaminases (ADAR) was discovered more than two decades ago. It provides additional regulation of animal and human transcriptome. In most cases, it occurs in nervous tissue where this results in conversion of adenosine to inosine at particular RNA sites. In the case of mRNA, an inosine residue is recognized by ribosome as guanine thus leading to amino acid substitutions during translation. Although such substitutions are shown to affect substantially functions of proteins (e.g. glutamate receptor) most of studies on RNA editing are mainly limited by analysis of nucleic acids even in the case of protein coding RNA transcripts. In this review, we propose the use of shotgun proteomics based on high resolution liquid chromatography and mass spectrometry for investigation of the effects of RNA editing at the protein level. Recently developed methods of big data processing allow combining the results of various omics techniques, being referred to as proteogenomics. The proposed proteogenomic approach for the analysis of RNA editing at the protein level is applicable for qualitative and quantitative analyses of protein edited sequences at the whole proteome level. Using this approach it will be possible to evaluate clinical importance of this phenomenon especially in the context of nervous system diseases.
Keywords: RNA-specific adenosine deaminases (ADAR); RNA editing; shotgun proteomics; proteogenomics

Cancer stem cell molecular markers verified in vivo by Y. S. Kim; A. M. Kaidina; J.-H. Chiang; K. N. Yarygin; A. Yu. Lupatov (43-54).
This systematic review was aimed at identification of cancer stem cells (CSC) molecular markers, which was validated in experiments on transplantation of tumor subpopulations with different phenotypes to immunodeficient animals. After careful examination of available papers, we selected 97 reports, fully consistent with the objectives of the study. The results of their analysis show that markers commonly used for CSC isolation include: CD133, СD44, ALDH, CD34, CD24, and EpCAM. The review also contains description of molecular features of some CSC markers; it also considers modern approaches to cancer treatment by targeting this population and evaluates perspectives of further development of the CSC theory.
Keywords: cancer stem cell; molecular markers; systematic review

Epigenetic markers of esophageal cancer: DNA methylation by O. I. Kit; D. I. Vodolazhskiy; E. N. Kolesnikov; N. N. Timoshkina (55-61).
Adenocarcinoma and squamous cell carcinoma are the most common types of esophageal malignant tumors. Their constant tendency to the increase in morbidity and high mortality rate underlie importance of the search for new biomarkers that complement and improve the early diagnosis of this disease. Despite significant efforts undertaken in this field the only marker of esophageal cancer, the ERBB2/HER2 status, is used in routine clinical practice. This review summarizes data on epigenetic markers characterizing aberrant methylation of the genome, which may be useful for early detection of esophageal cancer, prognosis and/or prediction of response to treatment. The development of new high-throughput technologies of genome-wide screening, such as BeadChip array and immunoprecipitation followed by sequencing, used not only for genome-wide genotyping, but also for the analysis of transcriptome and methylome, provides a comprehensive picture of genetic and epigenetic changes during tumorigenesis.
Keywords: esophageal cancer; methylation; biomarkers

Conformational polymorphysm of G-rich fragments of DNA Alu-repeats. I. Noncanonical structures by A. V. Sekridova; A. M. Varizhuk; O. N. Tatarinova; V. V. Severov; N. A. Barinov; I. P. Smirnov; V. N. Lazarev; D. V. Klinov; G. E. Pozmogova (62-71).
We report results of the first systematic study of conformational polymorphism of G-rich DNA fragments of Alu-repeats. Alu retrotransposons are primate-specific short interspersed elements. Using the Alu sequence of the prooncogen bcl2 intron and the consensus AluSx sequence as representative examples, we have determined characteristic Alu sites that are capable of adopting G-quadruplex (GQ) conformations (i.e., potential quadruplex sites—PQSAlu), and demonstrated by bioinformatics methods that these sites are Alu-specific in the human genome. Genomic frequencies of PQSAlu were assessed (~1/10000 bp). These sites were found to be characteristic of young (active) Alu families (Alu-Y). A recombinant DNA sequence bearing the Alu element of the human bcl2 gene (304 bp) and its PQS-mutant (Alu-PQS) were constructed. The formation of noncanonical structures in Alubcl2 dsDNA and their absence in the case of Alu-PQS have been shown using DMS-footprinting and atomic force microscopy (AFM). Expression vectors bearing wild-type and mutant Alu insertions in the promoter regions of the reporter gene have been prepared, and their regulatory effects have been compared during transfection of НЕК293 and HeLa cells. We suggest that the dynamic study of the spatial organization of Alu repeats may provide insight into the mechanisms of genomic rearrangements responsible for the development of many oncological and neurodegenerative diseases.
Keywords: G-quadruplex DNA; Alu-repeats; dynamics of DNA secondary structures

The role of neutrophil myeloperoxidase in the development of inflammation induced by thermal skin burns by E. V. Mikhalchik; L. I. Budkevich; Yu. A. Piterskaya; L. Yu. Penkov; T. S. Astamirova; N. V. Smolina; T. V. Vakhrusheva; O. M. Panasenko (72-75).
Luminol-dependent chemiluminescence (CL) of blood neutrophils stimulated with phorbol-12- myristate-13-acetate (PMA) and myeloperoxidase (MPO) activity of neutrophils and plasma have been investigated in children (n = 16) during the early period (1−7 days) after thermal skin burns exceeding 20% of total body surface. The CL level of stimulated neutrophils was higher in burn patients than in healthy children of the reference group (p lt; 0.01). Increased neutrophil MPO activity was found in 40% of patients, while increased plasma MPO activity was detected in 57% of patients. The albumin fraction isolated from plasma of burn patients increased the PMA-stimulated CL response of blood from healthy donors. These results suggest that the acute inflammatory response to the thermal burn causes neutrophil activation and MPO release into plasma. MPO-mediated modification of plasma proteins, particularly albumin, may stimulate neutrophil activation and provoke further inflammatory response of the body to the thermal injury.
Keywords: burns; chemiluminescence; neutrophils; myeloperoxidase; albumin

Caspase activity in lymphocytes of patients with depression and anxiety of different severity by A. A. Yakovlev; T. A. Druzhkova; M. N. Grishkina; A. B. Guekht; N. V. Gulyaeva (76-80).
Although borderline personality disorders (BPD) are most common psychiatric diseases, their pathogenesis still remains poorly understood. According to the literature and results of own previous studies, pathological changes associated with BPD may affect not only brain cells, but also cells of the immune system. One of the functionally important examples of communication between the immune and nervous systems in BPD is death of lymphocytes, particularly recognized in the most common mental disorder, depression. We have shown previously that activity of caspases, the key enzymes of programmed cell death increases in some types of BPD. In this study, we have investigated caspase activity in peripheral blood mononuclear cells (PBMC) of BPD patients of different severity. It has been found that in severe depression activity of initiator and executive caspases decreases as compared with control, and patients with a mild form of depression. In contrast, patients with severe form of anxiety were characterized by increased activity of the same enzymes as compared with control and patients with less severe forms of anxiety. Thus, the study of PBMC caspase can not only discriminate mild and severe forms of BPD, but can also help to identify specific molecular mechanisms responsible for the development of depression and anxiety.
Keywords: depression; anxiety; lymphocytes; apoptosis; caspases; borderline personality disorders

Features of blood-brain barrier formation affected by the modulation of HIF activity in astroglial and neuronal cells in vitro by V. A. Ruzaeva; A. V. Morgun; E. D. Khilazheva; N. V. Kuvacheva; E. A. Pozhilenkova; E. B. Boitsova; G. P. Martynova; T. E. Taranushenko; A. B. Salmina (81-86).
Barriergenesis is the process of maturation of the primary vascular network of the brain responsible for the establishment of the blood-brain barrier. It represents a combination of factors that, on the one hand, contribute to the process of migration and tubulogenesis of endothelial cells (angiogenesis), on the other hand, contribute to the formation of new connections between endothelial cells and other elements of the neurovascular unit. Astrocytes play a key role in barriergenesis, however, mechanisms of their action are still poorly examined. We have studied the effects of HIF-1 modulators acting on the cells of non-endothelial origin (neurons and astrocytes) on the development of the blood-brain barrier in vitro. Application of FM19G11 regulating expression of HIF-1 activity and GSI-1 suppressing gamma-secretase and/or proteasomal activity resulted in the elevated expression of thrombospondins and matrix metalloproteinases in the developing blood-brain barrier. However, it caused the opposite effect on VEGF expression thus promoting barrier maturation in vitro.
Keywords: blood-brain barrier; barriergenesis; neurovascular unit; angiogenesis; HIF-1 modulators