Biochemistry (Moscow) (v.76, #2)

Physiological scenarios of programmed loss of mitochondrial DNA function and death of yeast by S. A. Kochmak; D. A. Knorre; S. S. Sokolov; F. F. Severin (167-171).
Recently it was convincingly shown that the yeast Saccharomyces cerevisiae does possess the basic modules of programmed cell death machinery. As programmed cell death is suicide for a unicellular organism, it is reasonable to assume that they trigger the program when the death is beneficial for the rest of the population. Not surprisingly, most of the scenarios of physiological death of S. cerevisiae, i.e. cell death in stationary culture, during meiosis, during mating, and driven by viruses are dependent on quorum sensing, meaning that they depend on the cell density. Here we also discuss possible mechanisms that govern fitness decline during replicative aging of S. cerevisiae cells. We argue that loss of mitochondrial DNA function that occurs during replicative aging is programmed and adaptive. Indeed, yeast cells with nonfunctional mitochondrial DNA are known to be extremely stress-resistant, and also the presence of a subpopulation of such cells might protect the culture from degeneration by preventing the fixation of opportunistic mutations.
Keywords: aging; apoptosis; mitochondria; petite; yeast

Steered molecular dynamics simulation has revealed the mechanism of formate transport via the substrate channel of formate dehydrogenase. It is shown that the structural organization of the channel promotes the transport of formate anion in spite of the fact that the channel is too narrow even for such a small molecule. The conformational mobility of Arg284 residue, one of the residues forming the wall of the substrate channel, provides for the binding and delivery of formate to the active site.
Keywords: formate dehydrogenase; substrate channel; steered molecular dynamics

Molecular modeling of human lanosterol 14α-demethylase complexes with substrates and their derivatives by D. V. Mukha; S. I. Feranchuk; A. A. Gilep; S. A. Usanov (175-185).
Lanosterol 14α-demethylase (CYP51A1) is a key enzyme in sterol biosynthesis. In humans, this enzyme is involved in the cholesterol biosynthesis pathway. The majority of antifungal drugs are aimed at the inhibition of CYP51 in fungi. To elucidate the molecular mechanisms of highly specific protein-ligand recognition, we have developed a full-atomic model of human CYP51A1 and performed docking of natural substrates and their derivatives to the active site of the enzyme. The parameters of the binding enthalpy of substrates, intermediates, and final products of the reaction of 14α-demethylation were estimated using the MMPB(GB)SA algorithm. Dynamic properties and conformational changes of the protein globule upon binding of the ligand near the active site have been investigated by the molecular dynamics method. Our studies reveal that hydroxylated intermediate reaction products have a greater affinity than the initial substrates, which facilitates the multistage reaction without accumulation of intermediate products. The contribution to the free energy of steroid ligand binding of 30 amino acids forming the substrate-binding region of CYP51A1, as well as the influence of their substitutions to alanine on the stability of the protein molecule, has been clarified using alanine scanning modeling. We demonstrate that the most serious weakening of the binding is observed in the case of substitutions Y137A, F145A, V149A, I383A, and R388A. The results of molecular modeling are in agreement with the data obtained by analysis of primary sequences of representatives of the CYP51 family.
Keywords: CYP51A1; lanosterol 14α-demethylase; molecular modeling; docking; molecular dynamics; mutagenesis; alanine scanning

The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) is a component of the respiratory chain of various bacteria that generates a redox-driven transmembrane electrochemical Na+ potential. The Na+-NQR activity is known to be specifically inhibited by low concentrations of silver ions. Replacement of the conserved Cys377 residue with alanine in the NqrF subunit of Na+-NQR from Vibrio harveyi resulted in resistance of the enzyme to Ag+ and to other heavy metal ions. Analysis of the catalytic activity also showed that the rate of electron input into the mutant Na+-NQR decreased by about 14-fold in comparison to the wild type enzyme, whereas all other properties of NqrFC377A Na+-NQR including its stability remained unaffected.
Keywords: Na+-translocating NADH:quinone oxidoreductase; NqrF; ferredoxin:NADP+ oxidoreductase; sensitivity to heavy metals; protein stability

Interaction between MAK-V protein kinase and synaptopodin by S. V. Kalinichenko; P. N. Vikhreva; I. V. Korobko (196-201).
MAK-V protein kinase (also known as HUNK) was discovered more than decade ago but its functions and molecular mechanisms of action still remain mostly unknown. In an attempt to associate MAK-V with particular chains of molecular events, we searched for proteins interacting with the C-terminal domain of MAK-V protein kinase. We identified synaptopodin as a protein interaction partner for MAK-V and confirmed this interaction in various ways. Because synaptopodin is important for dendritic spine formation and plays a role in synaptic plasticity, our results might have significant impact on future studies for understanding the role of MAK-V in cells of the nervous system.
Keywords: MAK-V/HUNK protein kinase; synaptopodin; protein-protein interactions; brain

Toxin-binding proteins isolated from yellow mealworm Tenebrio molitor and wax moth Galleria mellonella by N. V. Bulushova; D. P. Zhuzhikov; L. I. Lyutikova; N. E. Kirillova; I. A. Zalunin; G. G. Chestukhina (202-208).
A 67-kDa protein that can specifically bind the activated Cry9A endotoxin under ligand-blotting conditions was purified from midgut epithelium apical membranes of wax moth Galleria mellonella by affinity chromatography. N-Terminal amino acid sequencing enabled identification of this protein as aminopeptidase N. In similar experiments, 66- and 58-kDa proteins specific to endotoxin Cry3A were isolated from the midgut epithelium apical membranes of Tenebrio molitor larvae. Mass spectrometry showed close similarity of the 58-kDa protein to the Tenebrio molitor α-amylase.
Keywords: δ-endotoxin; Bacillus thuringiensis ; Galleria mellonella ; Tenebrio molitor ; toxin-binding protein

A number of genetic or drug-induced pathophysiological disorders, particularly neurodegenerative diseases, have been reported to correlate with catalytic impairments of NADH:ubiquinone oxidoreductase (mitochondrial complex I). The vast majority of the data on catalytic properties of this energy-transducing enzyme have been accumulated from studies on bovine heart complex I preparations of different degrees of resolution, whereas almost nothing is known about the functional activities of the enzyme in neuronal tissues. Here a procedure for preparation of coupled inside-out submitochondrial particles from brain is described and their NADH oxidase activity is characterized. The basic characteristics of brain complex I, particularly the parameters of A/D-transition are found to be essentially the same as those previously reported for heart enzyme. The results show that coupled submitochondrial particles prepared from either heart or brain can equally be used as a model system for in vitro studies aimed to delineate neurodegenerative-associated defects of complex I.
Keywords: NADH:ubiquinone oxidoreductase; complex I; mitochondria; brain

In liver mitochondria fatty acids act as protonophoric uncouplers mainly with participation of internal membrane protein carriers — ADP/ATP and aspartate/glutamate antiporters. In this study the values of recoupling effects of carboxyatractylate and glutamate (or aspartate) were used to assess the degree of participation of ADP/ATP and aspartate/glutamate antiporters in uncoupling activity of fatty acids. These values were determined from the ability of these recoupling agents to suppress the respiration stimulated by fatty acids and to raise the membrane potential reduced by fatty acids. Increase in palmitic and lauric acid concentration was shown to increase the degree of participation of ADP/ATP antiporter and to decrease the degree of participation of aspartate/glutamate antiporter in uncoupling to the same extent. These data suggest that fatty acids are not only inducers of uncoupling of oxidative phosphorylation, but that they also act the regulators of this process. The linear dependence of carboxyatractylate and glutamate recoupling effects ratio on palmitic and lauric acids concentration was established. Comparison of the effects of fatty acids (palmitic, myristic, lauric, capric, and caprylic having 16, 14, 12, 10, and 8 carbon atoms, respectively) has shown that, as the hydrophobicity of fatty acids decreases, the effectiveness decreases to a greater degree than the respective values of their specific uncoupling activity. The action of fatty acids as regulators of uncoupling is supposed to consist of activation of transport of their anions from the internal to the external monolayer of the internal membrane with participation of ADP/ATP antiporter and, at the same time, in inhibition of this process with the participation of aspartate/glutamate antiporter.
Keywords: ADP/ATP antiporter; aspartate/glutamate antiporter; fatty acids; liver mitochondria; regulation; uncoupling

Bacterial proteins fold faster than eukaryotic proteins with simple folding kinetics by O. V. Galzitskaya; N. S. Bogatyreva; A. V. Glyakina (225-235).
Protein domain frequency and distribution among kingdoms was statistically analyzed using the SCOP structural database. It appeared that among chosen protein domains with the best resolution, eukaryotic proteins more often belong to α-helical and β-structural proteins, while proteins of bacterial origin belong to α/β structural class. Statistical analysis of folding rates of 73 proteins with known experimental data revealed that bacterial proteins with simple kinetics (23 proteins) exhibit a higher folding rate compared to eukaryotic proteins with simple folding kinetics (27 proteins). Analysis of protein domain amino acid composition showed that the frequency of amino acid residues in proteins of eukaryotic and bacterial origin is different for proteins with simple and complex folding kinetics.
Keywords: “all-or-none” transition; eukaryotic and bacterial proteins; folding intermediates; folding rate; protein folding

Prediction of folding nuclei in tRNA molecules by L. B. Pereyaslavets; M. V. Baranov; E. I. Leonova; O. V. Galzitskaya (236-244).
Prediction of folding nuclei in RNA molecules allows one to look in a new way at the problem of possible RNA base sequence folding and at problems associated with incorrect RNA folding, as well as at RNA structure stability. We have chosen a model and energy parameters for description of RNA structure. The algorithm for studying processes including protein folding/unfolding was successfully applied to calculations on tRNA. Four tRNA molecules were considered whose structures were obtained in the free state (tRNAPhe, tRNAAsp, tRNAfMet, and tRNALys). The calculated Φ-values for tRNA molecules correlate with experimental data showing that nucleotide residues in the D and T hairpin regions are involved in tRNA structure last, or more exactly, they are not included in the tRNA folding nucleus. High Φ-values in the anticodon hairpin region show that the nucleus of tRNA folding is localized just in that place.
Keywords: dynamic programming; folding nucleus; hydrogen bond; stacking and hydrophobic interactions; coarse-grained structural model; tRNA folding

A spectroscopic assay for detection of extrahelical thymine residues in DNA heteroduplexes under their modification by potassium permanganate has been developed. The assay is based on increase in absorbance at 420 nm due to accumulation of thymidine oxidation intermediates and soluble manganese dioxide. The analysis was carried out using a set of 19-bp DNA duplexes containing unpaired thymidines opposite tetrahydrofuranyl derivatives mimicking a widespread DNA damage (apurinic (AP) sites) and a library of 50-bp DNA duplexes containing all types of base mismatches in different surroundings. The relation between the selectivity of unpaired T oxidation and the thermal stability of DNA double helix was investigated. The method described here was shown to discriminate between DNA duplexes with one or two AP sites and to reveal thymine-containing mismatches and all noncanonical base pairs in AT-surroundings. Comparative results of CCM analysis and the rapid photometric assay for mismatch detection are demonstrated for the first time in the same model system. The chemical reactivity of target thymines was shown to correlate with local disturbance of double helix at the mismatch site. As the spectroscopic assay does not require the DNA cleavage reaction and gel electrophoresis, it can be easily automated and used for primary screening of somatic mutations.
Keywords: DNA structure; noncanonical pairs; heteroduplexes; chemical modification of heterocyclic bases; detection of mutations; apurinic sites

Peroxisome proliferator-activated receptor α (PPARα), one of the key ligand-activated nuclear receptors interacting with PPAR response elements (PPREs), may trigger the expression of PPAR-responsive genes and be involved in the transcriptional regulation of lipid metabolism, energy balance, and some diseases. Previous studies have demonstrated that the mouse Ppsig gene is a novel PPARα target gene taking a pivotal role in maintaining energy balance during fasting. Disparity between humans and rodents in their PPAR systems requires corroborating experiments to determine whether the hPpsig gene (Ppsig homologous gene in human) is also a PPARα target gene. In this work, eight putative PPREs in the promoter and first intron of hPpsig were identified. However, only one intronic PPRE could respond to PPARα by transient transfection. Furthermore, the binding activity of PPARα with this intronic PPRE was confirmed by electrophoretic mobility shift assay in vitro. This investigation might help to elucidate the transcriptional regulatory mechanisms of Ppsig in humans.
Keywords: EMSA; human Ppsig ; intronic PPREs; PPARα; transcriptional regulation

Exogenous thyroid hormones are regulators of cellular metabolism that involves, along with other cell structures, mitochondria. Mechanisms of the influence of thyroid hormones on the biogenesis of mtDNA are not fully understood due to their pleiotropic nature. Different ways of regulation of mitochondrial biogenesis by thyroid hormones are discussed in literature, but thyroid receptors, localized in both the nucleus and mitochondria, are the main elements of most pathways. Data on events occurring after receptor activation are rather contradictory. We investigated the degree of involvement of mitochondrial transcription factors in the biogenesis of mtDNA induced by triiodothyronine. The contribution of TFAM, TFB2M, and helicase Twinkle in thyroid-induced mtDNA biogenesis was assessed. The activation of TFAM and TFB2M expression is shown to be required for the induction of mtDNA biogenesis. The role of helicase Twinkle, the expression induction of which is also observed after triiodothyronine addition, remains unclear. The analysis of factors that activate TFAM and TFB2M expression showed that NRF-1 is the determinative regulator: deficiency of this factor leads to complete collapse of mtDNA biogenesis. However, lack of transcriptional coactivator PGC-1α did not lead to significant reduction in thyroid-induced biogenesis, whereas literature data point to its key role in the biogenesis of mitochondria. Thus, in this study the role of key transcription factors in mtDNA biogenesis induced by triiodothyronine was demonstrated for the first time in a model system.
Keywords: biogenesis; mtDNA; thyroid hormones; transcription factors; triiodothyronine

Oxidation of glyceraldehyde-3-phosphate dehydrogenase decreases sperm motility by Yu. L. Elkina; M. M. Atroshchenko; E. E. Bragina; V. I. Muronetz; E. V. Schmalhausen (268-272).
The relation between the activity of the sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) and the motility of sperms was investigated. It was found that the mean value of GAPDS activity in sperm samples with low motility is 2.5–3-fold lower than that in samples with high motility. Sperm motility was shown to diminish in the presence of superoxide anion, hydroxyl radical, and hydrogen peroxide. The decrease in sperm motility in the presence of hydrogen peroxide was proportional to the concentration of the oxidant and correlated with the decrease in GAPDS activity (r = 0.96). Based on the literature data on the importance of GAPDS for the motility of sperms together with the presented observations, it was concluded that the decrease in the sperm motility in the presence of reactive oxygen species is due to the oxidation of GAPDS and inhibition of glycolysis.
Keywords: glyceraldehyde-3-phosphate dehydrogenase; motility; oxidation; reactive oxygen species; spermatozoa

Kinetic mechanism of human apurinic/apyrimidinic endonuclease action in nucleotide incision repair by N. A. Timofeyeva; V. V. Koval; A. A. Ishchenko; M. K. Saparbaev; O. S. Fedorova (273-281).
Human major apurinic/apyrimidinic endonuclease (APE1) is a multifunctional enzyme that plays a central role in DNA repair through the base excision repair (BER) pathway. Besides BER, APE1 is involved in an alternative nucleotide incision repair (NIR) pathway that bypasses glycosylases. We have analyzed the conformational dynamics and the kinetic mechanism of APE1 action in the NIR pathway. For this purpose we recorded changes in the intensity of fluorescence of 2-aminopurine located in two different positions in a substrate containing dihydrouridine (DHU) during the interaction of the substrate with the enzyme. The enzyme was found to change its conformation within the complex with substrate and also within the complex with the reaction product, and the release of the enzyme from the complex with the product seemed to be the limiting stage of the enzymatic process. The rate constants of the catalytic cleavage of DHU-containing substrates by APE1 were comparable with the appropriate rate constants for substrates containing apurinic/apyrimidinic site or tetrahydrofuran residue, which suggests that NIR is a biologically important process.
Keywords: APE1; nucleotide incision repair (NIR); kinetic mechanism