Biochemistry (Moscow) (v.75, #13)

Ribosomal tunnel and translation regulation by A. A. Bogdanov; N. V. Sumbatyan; A. V. Shishkina; V. V. Karpenko; G. A. Korshunova (1501-1516).
This review describes the results of recent studies of the ribosomal tunnel (RT), the major function of which is to allow the smooth passage of nascent polypeptides with different sequences from the peptidyl transferase center of the ribosome to the tunnel exit, where the folding of protein molecules begins. The features of structural organization of RT and their role in modulation and stabilization of the nascent chain conformation are discussed. Structural features of macrolide binding sites as well as application of macrolide antibiotics and their derivatives as tools to investigate ligand-tunnel wall interactions are also considered. Several examples of strong and specific interactions of regulatory polypeptides with nucleotide and amino acid residues of RT that lead to ribosome stalling and translational arrest are described in detail. The role of these events in regulation of expression of certain genes is discussed on the basis of recent high-resolution structural studies of nascent chains in the RT.
Keywords: ribosome; ribosomal tunnel; growing polypeptide chain; macrolides; leader peptides; translation regulation; cation-π interactions

This review analyzes the concept according to which the pathway of synthesized peptide from the ribosome peptidyl transferase center to the exit domain goes along the tunnel of the large subparticle. Experimental data on the accessibility of the nascent polypeptide chain to molecules of modifying agents and fluorescence quenchers are considered. Results of localization of the exit site for the nascent peptide on the ribosome surface, possible conformational states of the peptide, and its mobility and folding on the ribosome are analyzed. The analysis is based on the ribosomal tunnel parameters obtained using X-ray crystallography of whole ribosomes and large ribosomal subparticles. Special attention is given to data that do not fit in the concept of the “tunnel for peptide exit“ and to results already obtained before the reliable tunnel visualization using X-ray crystallography was achieved.
Keywords: nascent polypeptide; ribosomal tunnel; nascent polypeptide chain exit domain; nascent polypeptide accessibility

14-3-3 Proteins and regulation of cytoskeleton by N. N. Sluchanko; N. B. Gusev (1528-1546).
The proteins of the 14-3-3 family are universal adapters participating in multiple processes running in the cell. We describe the structure, isoform composition, and distribution of 14-3-3 proteins in different tissues. Different elements of 14-3-3 structure important for dimer formation and recognition of protein targets are analyzed in detail. Special attention is paid to analysis of posttranslational modifications playing important roles in regulation of 14-3-3 function. The data of the literature concerning participation of 14-3-3 in regulation of intercellular contacts and different elements of cytoskeleton formed by microfilaments are analyzed. We also describe participation of 14-3-3 in regulation of small G-proteins and protein kinases important for proper functioning of cytoskeleton. The data on the interaction of 14-3-3 with different components of microtubules are presented, and the probable role of 14-3-3 in developing of certain neurodegenerative diseases is discussed. The data of the literature concerning the role of 14-3-3 in formation and normal functioning of intermediate filaments are also reviewed. It is concluded that due to its adapter properties 14-3-3 plays an important role in cytoskeleton regulation. The cytoskeletal proteins that are abundant in the cell might compete with the other protein targets of 14-3-3 and therefore can indirectly regulate many intracellular processes that are dependent on 14-3-3.
Keywords: 14-3-3; phosphorylation; cytoskeleton

The basic mechanisms are considered that are responsible for producing biochemical polymorphism of human proteins realized at three basic levels: the structures of genome and genes; the transcription and maturation of transcripts; the postsynthetic formation of functionally active protein products of gene expression. The data on biochemical polymorphism of growth hormone (GH) and some other proteins that are directly or indirectly necessary for its functioning and support this polymorphism by polylocus, polyallelism, alternative splicing, and various postsynthetic modifications are analyzed. The role of polymorphic proteins of the GH system is discussed in formation of a variety of oligomeric molecular structures of this system (multicomponent transport complexes, receptors, and endocellular protein ensembles involved in the regulation of gene expression). It is emphasized that such structural polymorphism significantly influences the biological effects in various parts of the GH system during physiological processes and in tumors, in particular in prostate cancer.
Keywords: biochemical polymorphism; growth hormone system; prostate cancer

Telomerase: Structure, functions, and activity regulation by M. I. Zvereva; D. M. Shcherbakova; O. A. Dontsova (1563-1583).
Telomerase is the enzyme responsible for maintenance of the length of telomeres by addition of guanine-rich repetitive sequences. Telomerase activity is exhibited in gametes and stem and tumor cells. In human somatic cells proliferation potential is strictly limited and senescence follows approximately 50–70 cell divisions. In most tumor cells, on the contrary, replication potential is unlimited. The key role in this process of the system of the telomere length maintenance with involvement of telomerase is still poorly studied. No doubt, DNA polymerase is not capable to completely copy DNA at the very ends of chromosomes; therefore, approximately 50 nucleotides are lost during each cell cycle, which results in gradual telomere length shortening. Critically short telomeres cause senescence, following crisis, and cell death. However, in tumor cells the system of telomere length maintenance is activated. Besides catalytic telomere elongation, independent telomerase functions can be also involved in cell cycle regulation. Inhibition of the telomerase catalytic function and resulting cessation of telomere length maintenance will help in restriction of tumor cell replication potential. On the other hand, formation of temporarily active enzyme via its intracellular activation or due to stimulation of expression of telomerase components will result in telomerase activation and telomere elongation that can be used for correction of degenerative changes. Data on telomerase structure and function are summarized in this review, and they are compared for evolutionarily remote organisms. Problems of telomerase activity measurement and modulation by enzyme inhibitors or activators are considered as well.
Keywords: telomerase; senescence; TERT; telomerase RNA; telomerase reaction cycle; telomerase activity; telomerase regulators

Generation of recombinant antibodies and means for increasing their affinity by E. P. Altshuler; D. V. Serebryanaya; A. G. Katrukha (1584-1605).
Highly specific interaction with foreign molecules is a unique feature of antibodies. Since 1975, when Keller and Milstein proposed the method of hybridoma technology and prepared mouse monoclonal antibodies, many antibodies specific to various antigens have been obtained. Recent development of methods for preparation of recombinant DNA libraries and in silico bioinformatics approaches for protein structure analysis makes possible antibody preparation using gene engineering approaches. The development of gene engineering methods allowed creating recombinant antibodies and improving characteristics of existing antibodies; this significantly extends the applicability of antibodies. By modifying biochemical and immunochemical properties of antibodies by changing their amino acid sequences it is possible to create antibodies with properties optimal for certain tasks. For example, application of recombinant technologies resulted in antibody preparation of high affinity significantly exceeding the initial affinity of natural antibodies. In this review we summarize information about the structure, modes of preparation, and application of recombinant antibodies and their fragments and also consider the main approaches used to increase antibody affinity.
Keywords: recombinant antibody fragment; affinity; maturation; modeling; in vitro evolution; display

Cross-linked nucleic acids: isolation, structure, and biological role by V. A. Efimov; S. V. Fedyunin (1606-1627).
This review includes literature data on main types of reagents inducing covalent cross-links of nucleic acids. Reactivity of cross-linking agents, preferable sites for their binding, and methods for determination of location of cross-links in duplex are discussed. Biological responses of cells to cross-linking in nucleic acids, i.e. replication and transcription blocking, onset of repair processes, and apoptotic cell death are considered, as well as application of cross-linking reagents as medicinal drugs for solving molecular-biological problems.
Keywords: nucleic acids; cross-link; cross-linking agent

Multiparametric determination of genes and their point mutations for identification of beta-lactamases by M. Yu. Rubtsova; M. M. Ulyashova; T. T. Bachmann; R. D. Schmid; A. M. Egorov (1628-1649).
More than half of all currently used antibiotics belong to the beta-lactam group, but their clinical effectiveness is severely limited by antibiotic resistance of microorganisms that are the causative agents of infectious diseases. Several mechanisms for the resistance of Enterobacteriaceae have been established, but the main one is the enzymatic hydrolysis of the antibiotic by specific enzymes called beta-lactamases. Beta-lactamases represent a large group of genetically and function-ally different enzymes of which extended-spectrum beta-lactamases (ESBLs) pose the greatest threat. Due to the plasmid localization of the encoded genes, the distribution of these enzymes among the pathogens increases every year. Among ESBLs the most widespread and clinically relevant are class A ESBLs of TEM, SHV, and CTX-M types. TEM and SHV type ESBLs are derived from penicillinases TEM-1, TEM-2, and SHV-1 and are characterized by several single amino acid substitutions. The extended spectrum of substrate specificity for CTX-M beta-lactamases is also associated with the emergence of single mutations in the coding genes. The present review describes various molecular-biological methods used to identify determinants of antibiotic resistance. Particular attention is given to the method of hybridization analysis on microarrays, which allows simultaneous multiparametric determination of many genes and point mutations in them. A separate chapter deals with the use of hybridization analysis on microarrays for genotyping of the major clinically significant ESBLs. Specificity of mutation detection by means of hybridization analysis with different detection techniques is compared.
Keywords: antibiotic resistance; beta-lactamases; gene determination; single nucleotide polymorphism; hybridization analysis; microarrays

Acute myeloid leukemia (AML) is a malignant blood disease caused by different mutations that enhance the pro-liferative activity and survival of blood cells and affect their differentiation and apoptosis. The most frequent disorders in AML are translocations between chromosomes 21 and 8 leading to production of a chimeric oncogene, AML1-ETO, and hyperexpression of the receptor tyrosine kinase KIT. Mutations in these genes often occur jointly. The presence in cells of two activated oncogenes is likely to trigger their malignization. The current approaches for treatment of oncologic diseases (bone marrow transplantation, radiotherapy, and chemotherapy) have significant shortcomings, and thus many laboratories are intensively developing new approaches against leukemias. Inhibiting expression of activated leukemic oncogenes based on the principle of RNA interference seems to be a promising approach in this field.
Keywords: acute myeloid leukemia (AML); leukemic oncogenes; AML1-ETO ; c-kit ; RNA interference

This paper reviews chromosomal abnormalities observed in oncological diseases, the history of discovery of chromosomal translocations (a widespread type of chromosomal abnormalities), and statistical data showing a correlation between translocations and emergence of oncological diseases (in particular leukemia). The importance of detection of minimal residual disease (MRD) in treatment of leukemia associated with translocations is discussed along with methods of MRD diagnosis, followed by description of a novel diagnostic procedure for the detection of single chimeric mRNA molecules serving as MRD markers. This procedure includes a number of improvements, of which the most important is the use of a PCR version of the method of nanocolonies (other names are molecular colonies, polonies) that provides for the determination of the absolute titer of RNA tumor markers, excludes false positive results in the detection of chimeric molecules, and significantly exceeds other methods in the sensitivity of MRD detection.
Keywords: chromosomal translocation; leukemia; minimal residual disease; RT-PCR; nanocolonies; molecular colonies; polonies; PCR colonies

Constitutive and induced functions of the p53 gene by A. O. Zheltukhin; P. M. Chumakov (1692-1721).
The p53 tumor suppressor serves to secure genetic stability of multicellular organisms. It suppresses the accumulation of mutations in somatic cells and substantially decreases the probability of malignant diseases. The p53 gene acts highly selectively through multiple mechanisms. Under relatively favorable conditions, p53 helps to maintain intracellular homeostasis by balancing anabolic and catabolic processes and by timely elimination of reactive oxygen species. These functions of p53 facilitate maximal efficiency and survival of cells under conditions of physiological stresses. In the case of grave molecular damage caused by severe stress, a significant amount of highly active p53 is induced leading to irreversible growth arrest and programmed cell death. The induced functions of p53 secure the timely elimination from the organism of damaged and potentially dangerous cells. Collectively, the functions of p53 contribute to the prevention of malignant and other diseases and decelerate the aging process.
Keywords: p53 tumor suppressor; cancer prevention; signal transduction; molecular damage; aging; apoptosis; autophagy; cell cycle control; oncogenes; tumor suppressor genes