Biochemistry (Moscow) (v.83, #2)

Activation of Yeast Mitochondrial Translation: Who Is in Charge? by K. S. Derbikova; S. A. Levitsky; I. V. Chicherin; E. N. Vinogradova; P. A. Kamenski (87-97).
Mitochondrial genome has undergone significant reduction in a course of evolution; however, it still contains a set of protein-encoding genes and requires translational machinery for their expression. Mitochondrial translation is of the prokaryotic type with several remarkable differences. This review is dedicated to one of the most puzzling features of mitochondrial protein synthesis, namely, the system of translational activators, i.e., proteins that specifically regulate translation of individual mitochondrial mRNAs and couple protein biosynthesis with the assembly of mitochondrial respiratory chain complexes. The review does not claim to be a comprehensive analysis of all published data; it is rather focused on the idea of the “core component” of the translational activator system.
Keywords: mitochondria; ribosomes; translation; mRNA; translational activators; complex assembly

Retrograde Signaling as a Mechanism of Yeast Adaptation to Unfavorable Factors by T. A. Trendeleva; R. A. Zvyagilskaya (98-106).
Mitochondria perform many essential functions in eukaryotic cells. Being the main producers of ATP and the site of many catabolic and anabolic reactions, they participate in intracellular signaling, proliferation, aging, and formation of reactive oxygen species. Mitochondrial dysfunction is the cause of many diseases and even cell death. The functioning of mitochondria in vivo is impossible without interaction with other cellular compartments. Mitochondrial retrograde signaling is a signaling pathway connecting mitochondria and the nucleus. The major signal transducers in the yeast retrograde response are Rtg1p, Rtg2p, and Rtg3p proteins, as well as four additional negative regulatory factors–Mks1p, Lst8p, and two 14-3-3 proteins (Bmh1/2p). In this review, we analyze current information on the retrograde signaling in yeast that is regarded as a stress or homeostatic response mechanism to changes in various metabolic and biosynthetic activities that occur upon mitochondrial dysfunction. We also discuss relations between retrograde signaling and other signaling pathways in the cell.
Keywords: retrograde signaling; yeast; TOR; RTG; autophagy; alternative oxidase; pleiotropic drug resistance

Multiple myeloma nephropathy occurs due to the aggregate formation by monoclonal immunoglobulin light chains (Bence-Jones proteins) in kidneys of patients with multiple myeloma. The mechanism of amyloid deposit formation is still unclear. Earlier, the key role in the fibril formation has been assigned to the variable domains that acquired amyloidogenic properties as a result of somatic mutations. However, fibril formation by the Bence-Jones protein BIF was found to be the function of its constant domain. The substitution of Ser177 by Asn in the constant domain of the BIF protein is most likely an inherited than a somatic mutation. To study the role of this mutation in amyloidogenesis, the recombinant Bence-Jones protein BIF and its mutant with the N177S substitution typical for the known immunoglobulin Cκ allotypes Km1, Km1,2, and Km3 were isolated. The morphology of aggregates formed by the recombinant proteins under conditions similar to those occurring during the protein transport in bloodstream and its filtration into the renal glomerulus, in the distal tubules, and in the proximal renal tubules was analyzed by atomic force microscopy. The nature of the aggregates formed by BIF and its N177S mutant during incubation for 14 days at 37°C strongly differed and depended on both pH and the presence of a reducing agent. BIF formed fibrils at pH 7.2, 6.5, and 10.1, while the N177S mutant formed fibrils only at alkaline pH 10.1. The refolding of both proteins in the presence of 5 mM dithiothreitol resulted in the formation of branched structures.
Keywords: Bence-Jones proteins; myeloma; aggregation; atomic force microscopy

The kinetic mechanism of the interaction of nonsteroidal anti-inflammatory drugs (NSAIDs) with their main pharmacological target, prostaglandin H synthase (PGHS), has not yet been established. We showed that inhibition of PGHS-1 from sheep vesicular glands by naproxen (a representative of NSAIDs) demonstrates a non-competitive character with respect to arachidonic acid and cannot be described within a framework of the commonly used kinetic schemes. However, it can be described by taking into account the negative cooperativity of naproxen binding to the cyclooxygenase active sites of the PGHS-1 homodimer (the first naproxen molecule forms a more stable complex (K 1 = 0.1 μM) with the enzyme than the second naproxen molecule (K 2 = 9.2 μM)). An apparent non-competitive interaction of PGHS-1 with naproxen is due to slow dissociation of the enzyme–inhibitor complexes. The same experimental data could also be described using commonly accepted kinetic schemes, assuming that naproxen interacts was a mixture of two enzyme species with the inhibition constants K α = 0.05 μM and K β = 18.3 μM. Theoretical analysis and numerical calculations show that the phenomenon of kinetic convergence of these two models has a general nature: when K 2 >> K1, the kinetic patterns (for transient kinetics and equilibrium state) generated by the cooperative model could be described by a scheme assuming the presence of two enzyme forms with the inhibition constants K α = K 1/2, K β = 2·K 2. When K 2<< K 1, the cooperative model can be presented as a scheme with two inhibitor molecules simultaneously binding to the enzyme with the observed inhibition constant K (K = K 1·K 2). The assumption on the heterogeneity of the enzyme preparation in relation to its affinity to the inhibitor can be used instead of the assumption on the negative cooperativity of the enzyme–inhibitor interactions for convenient and easy practical description of such phenomena in enzymology, biotechnology, pharmacology, and other fields of science.
Keywords: PGHS-1; COX-1; cyclooxygenase; NSAIDs; inhibitors; naproxen; Aleve; kinetics; cooperativity; homodimer

Comparison of Methods of Detection of Exceptional Sequences in Prokaryotic Genomes by I. S. Rusinov; A. S. Ershova; A. S. Karyagina; S. A. Spirin; A. V. Alexeevski (129-139).
Many proteins need recognition of specific DNA sequences for functioning. The number of recognition sites and their distribution along the DNA might be of biological importance. For example, the number of restriction sites is often reduced in prokaryotic and phage genomes to decrease the probability of DNA cleavage by restriction endonucleases. We call a sequence an exceptional one if its frequency in a genome significantly differs from one predicted by some mathematical model. An exceptional sequence could be either under- or over-represented, depending on its frequency in comparison with the predicted one. Exceptional sequences could be considered biologically meaningful, for example, as targets of DNA-binding proteins or as parts of abundant repetitive elements. Several methods to predict frequency of a short sequence in a genome, based on actual frequencies of certain its subsequences, are used. The most popular are methods based on Markov chain models. But any rigorous comparison of the methods has not previously been performed. We compared three methods for the prediction of short sequence frequencies: the maximum-order Markov chain model-based method, the method that uses geometric mean of extended Markovian estimates, and the method that utilizes frequencies of all subsequences including discontiguous ones. We applied them to restriction sites in complete genomes of 2500 prokaryotic species and demonstrated that the results depend greatly on the method used: lists of 5% of the most under-represented sites differed by up to 50%. The method designed by Burge and coauthors in 1992, which utilizes all subsequences of the sequence, showed a higher precision than the other two methods both on prokaryotic genomes and randomly generated sequences after computational imitation of selective pressure. We propose this method as the first choice for detection of exceptional sequences in prokaryotic genomes.
Keywords: DNA sequence; prokaryotic genome; compositional bias; Markov chain model; restriction–modification system; restriction sites

Comparative Action of Cardiotonic Steroids on Intracellular Processes in Rat Cortical Neurons by A. V. Lopachev; O. M. Lopacheva; K. A. Nikiforova; I. S. Filimonov; T. N. Fedorova; E. E. Akkuratov (140-151).
Binding to Na+,K+-ATPase, cardiotonic steroids (CTS) activate intracellular signaling cascades that affect gene expression and regulation of proliferation and apoptosis in cells. Ouabain is the main CTS used for studying these processes. The effects of other CTS on nervous tissue are practically uncharacterized. Previously, we have shown that ouabain affects the activation of mitogen-activated protein kinases (MAP kinases) ERK1/2, p38, and JNK. In this study, we compared the effects of digoxin and bufalin, which belong to different subclasses of CTS, on primary culture of rat cortical cells. We found that CTS toxicity is not directly related to the degree of Na+,K+-ATPase inhibition, and that bufalin and digoxin, like ouabain, are capable of activating ERK1/2 and p38, but with different concentration and time profiles. Unlike bufalin and ouabain, digoxin did not decrease JNK activation after long-term incubation. We concluded that the toxic effect of CTS in concentrations that inhibit less than 80% of Na+,K+-ATPase activity is related to ERK1/2 activation as well as the complex profile of MAP kinase activation. A direct correlation between Na+,K+-ATPase inhibition and the degree of MAP kinase activation is only observed for ERK1/2. The different action of the three CTS on JNK and p38 activation may indicate that it is associated with intracellular signaling cascades triggered by protein–protein interactions between Na+,K+-ATPase and various partner proteins. Activation of MAP kinase pathways by these CTS occurs at concentrations that inhibit Na+,K+-ATPase containing the α1 subunit, suggesting that these signaling cascades are realized via α1. The results show that the signaling processes in neurons caused by CTS can differ not only because of different inhibitory constants for Na+,K+-ATPase.
Keywords: cardiotonic steroids; digoxin; bufalin; ouabain; Na+,K+-ATPase; primary cultures of neurons; toxicity; MAP kinases; ERK1/2; p38; JNK

2,5-Diketopiperazines: A New Class of Poly(ADP-ribose)polymerase Inhibitors by D. K. Nilov; K. I. Yashina; I. V. Gushchina; A. L. Zakharenko; M. V. Sukhanova; O. I. Lavrik; V. K. Švedas (152-158).
We show for the first time that natural 2,5-diketopiperazines (cyclic dipeptides) can suppress the activity of the important anticancer target poly(ADP-ribose)polymerase (PARP). Cyclo(L-Ala-L-Ala) and cyclo(L-Ala-D-Ala) can interact with the key residues of the PARP-1 active site, as demonstrated using docking and molecular dynamics simulations. One of the amide groups of cyclo(L-Ala-L-Ala) and cyclo(L-Ala-D-Ala) forms hydrogen bonds with the Gly863 residue, while the second amide group can form a hydrogen bond with the catalytic residue Glu988, and the side chain can make a hydrophobic contact with Ala898. Newly identified diketopiperazine inhibitors are promising basic structures for the design of more effective inhibitors of PARP family enzymes. The piperazine core with two chiral centers provides many opportunities for structural optimization.
Keywords: diketopiperazine; piperazinedione; poly(ADP-ribose)polymerase; inhibitor; molecular modeling; docking; molecular dynamics

One of the important components of the concept of aging-phenoptosis (programmed aging) is the notion of aging as an accelerator of evolution having the rank of subconcept. For many reasons, the main being the problematic experimental testing of evolutionary hypotheses, verification of the above-mentioned subconcept can be based primarily on analysis of the internal inconsistency of heuristic models and their correspondence to undisputedly observed facts. To illustrate the acceleration mechanism, and most importantly to structure the evolutionary process in communities that include naturally weakened individuals, V. P. Skulachev offered in 2003 a conceptual model that he later called a “fable about hares”. Despite its simplicity, this model has undoubted internal logic. The natural trend in the development of conceptual models is their translation into the language of mathematics. The purpose of the present work was to create a variation of the known multi-agent model “predator–prey” that would allow us to “see” how the presence in the prey population of naturally weakened (old) members stimulates the selection of individuals with traits whose adaptive potential is not devaluated with age. The model (http://homebear.ru/PD) was developed on the Java platform, version 6, NetBeans development environment 8.2. Statistical analysis and preparation of illustrative materials were carried out using environment R, version 3.4.1. The results of numerical experiments set using our model correspond in principle to the provisions of the heuristic model of Skulachev and, consequently, confirm the absence in it of logical contradictions.
Keywords: aging; phenoptosis; multi-agent modeling; evolution; fable about hares

In this review, we shortly summarize the data of our studies (and also corresponding studies of other authors) on the new mechanism of myoglobin (Mb) deoxygenation in a cell, according to which Mb acts as an oxygen transporter, and its affinity for the ligand, like in other transporting proteins, is regulated by the interaction with the target, in our case, mitochondria (Mch). We firstly found that contrary to previously formulated and commonly accepted concepts, oxymyoglobin (MbO2) deoxygenation occurs only via interaction of the protein with respiring mitochondria (low $${p_{{O_2}}}$$ p O 2 values are necessary but not sufficient for this process to proceed). Detailed studies of the mechanism of Mb–Mch interaction by various physicochemical methods using natural and artificial bilayer phospholipid membranes showed that: (i) the rate of MbO2 deoxygenation in the presence of respiring Mch fully coincides with the rate of O2 uptake by mitochondria from a solution irrespectively of their state (native coupled, freshly frozen, or FCCP-uncoupled), i.e. it is determined by the respiratory activity of Mch; (ii) Mb nonspecifically binds to membrane phospholipids of the outer mitochondrial membrane, while any Mb-specific protein or phospholipid sites on it are lacking; (iii) oxygen uptake by Mch from a solution and the uptake of Mb-bound oxygen are two different processes, as their rates are differently affected by proteins (e.g. lysozyme) that compete with MbO2 for binding to the mitochondrial membrane; (iv) electrostatic forces significantly contribute to the Mb–membrane interactions; the dependence of these interactions on ionic strength is provided by the local electrostatic interactions between anionic groups of phospholipids (the heads) and invariant Lys and Arg residues near the Mb heme pocket; (v) interactions of Mb with phospholipid membranes promote conformational changes in the protein, primarily in its heme pocket, without significant alterations in the protein secondary and tertiary structures; and (vi) Mb–membrane interactions lead to decrease in the affinity of myoglobin for O2, which could be monitored by the increase in the MbO2 autooxidation rate under aerobic conditions and under anaerobic ones, by the shift in the MbO2/Mb(2) equilibrium towards the ligand-free protein. The decrease in the affinity of Mb for the ligand should facilitate O2 dissociation from MbO2 at physiological $${p_{{O_2}}}$$ p O 2 values in cells.
Keywords: myoglobin; mitochondria; spatial structure; deoxygenation mechanism