Biochemistry (Moscow) (v.77, #3)
Cys2His2 zinc finger protein family: Classification, functions, and major members by S. V. Razin; V. V. Borunova; O. G. Maksimenko; O. L. Kantidze (217-226).
Cys2His2 (C2H2)-type zinc fingers are widespread DNA binding motifs in eukaryotic transcription factors. Zinc fingers are short protein motifs composed of two or three β-layers and one α-helix. Two cysteine and two histidine residues located in certain positions bind zinc to stabilize the structure. Four other amino acid residues localized in specific positions in the N-terminal region of the α-helix participate in DNA binding by interacting with hydrogen donors and acceptors exposed in the DNA major groove. The number of zinc fingers in a single protein can vary over a wide range, thus enabling variability of target DNA sequences. Besides DNA binding, zinc fingers can also provide protein-protein and RNA-protein interactions. For the most part, proteins containing the C2H2-type zinc fingers are trans regulators of gene expression that play an important role in cellular processes such as development, differentiation, and suppression of malignant cell transformation (oncosuppression).
Keywords: zinc finger; DNA-binding protein; transcription; regulation of gene expression
ERBB oncogene proteins as targets for monoclonal antibodies by O. L. Polanovski; E. N. Lebedenko; S. M. Deyev (227-245).
General properties of the family of tyrosine kinase ERBB receptors are considered in connection with their role in the generation of cascades of signal transduction in normal and tumor cells. Causes of acquisition of oncogene features by genes encoding these receptors and their role in tumorigenesis are analyzed. Anti-ERBB monoclonal antibodies approved for therapy are described in detail, and mechanisms of their antitumor activity and development of resistance to them are reviewed. The existing and the most promising strategies for creating and using monoclonal antibodies and their derivatives for therapy of cancer are discussed.
Keywords: EGFR; HER2/neu; receptor tyrosine kinases ERBB1-4; signaling cascades; Herceptin
Functional phenotypes of macrophages and the M1-M2 polarization concept. Part I. Proinflammatory phenotype by Y. Sh. Schwartz; A. V. Svistelnik (246-260).
Current concepts concerning the main functional phenotypes of mononuclear phagocytes are systematized, molecular mechanisms of their formation are considered, and the functional polarization concept of macrophages is critically analyzed. Mechanisms of macrophage priming activation mediated by pattern recognition receptors TLR, NLR, RLR, and CLR are described, and the features of each phenotype acquired via various pattern recognition receptors are emphasized. It is concluded that there is a huge variety of proinflammatory phenotypes from highly to poorly polarized ones. Thus the widespread notion of “classical activation” of macrophage concerns just a particular case of proinflammatory phenotype formation.
Keywords: macrophage; polarization; phenotype; TLR; NLR; RLR; CLR
E2F1 enhances 8-Chloro-adenosine-induced G2/M arrest and apoptosis in A549 and H1299 lung cancer cells by Hong-Ying Duan; Ji-Xiang Cao; Jun-Juan Qi; Guo-Sheng Wu; Shu-Yan Li; Guo-Shun An; Hong-Ti Jia; Wang-Wei Cai; Ju-Hua Ni (261-269).
The E2F1 transcription factor is a well known regulator of cell proliferation and apoptosis, but its role in response to DNA damage is less clear. 8-Chloro-adenosine (8-Cl-Ado), a nucleoside analog, can inhibit proliferation in a variety of human tumor cells. However, it is still elusive how the agent acts on tumors. Here we show that A549 and H1299 cells formed DNA double-strand breaks after 8-Cl-Ado exposure, accompanied by E2F1 upregulation at protein level. Overexpressed wild-type (E2F1-wt) colocalized with double-strand break marker γ-H2AX and promoted G2/M arrest in 8-Cl-Ado-exposed A549 and H1299, while expressed S31A mutant of E2F1 (E2F1-mu) significantly reduced ability to accumulate at sites of DNA damage and G2/M arrest, suggesting that E2F1 is required for activating G2/M checkpoint pathway upon DNA damage. Transfection of either E2F1-wt or E2F1-mu plasmid promoted apoptosis in 8-Cl-Ado-exposed cells, indicating that 8-Cl-Ado may induce apoptosis in E2F1-dependent and E2F1-independent ways. These findings demonstrate that E2F1 plays a crucial role in 8-Cl-Ado-induced G2/M arrest but is dispensable for 8-Cl-Ado-induced apoptosis. These data also suggest that the mechanism of 8-Cl-Ado action is complicated.
Keywords: E2F1; 8-chloro-adenosine; DNA double-stranded breaks; G2/M arrest; apoptosis
Initiation of 8-oxoguanine base excision repair within trinucleotide tandem repeats by A. G. Derevyanko; A. V. Endutkin; A. A. Ishchenko; M. K. Saparbaev; D. O. Zharkov (270-279).
Trinucleotide repeat expansion provides a molecular basis for several devastating neurodegenerative diseases. In particular, expansion of a CAG run in the human HTT gene causes Huntington’s disease. One of the main reasons for triplet repeat expansion in somatic cells is base excision repair (BER), involving damaged base excision and repair DNA synthesis that may be accompanied by expansion of the repaired strand due to formation of noncanonical DNA structures. We have analyzed the kinetics of excision of a ubiquitously found oxidized purine base, 8-oxoguanine (oxoG), by DNA glycosylase OGG1 from the substrates containing a CAG run flanked by AT-rich sequences. The values of k 2 rate constant for the removal of oxoG from triplets in the middle of the run were higher than for oxoG at the flanks of the run. The value of k 3 rate constant dropped starting from the third CAG-triplet in the run and remained stable until the 3′-terminal triplet, where it decreased even more. In nuclear extracts, the profile of oxoG removal rate along the run resembled the profile of k 2 constant, suggesting that the reaction rate in the extracts is limited by base excision. The fully reconstituted BER was efficient with all substrates unless oxoG was near the 3′-flank of the run, interfering with the initiation of the repair. DNA polymerase β was able to perform a strand-displacement DNA synthesis, which may be important for CAG run expansion initiated by BER.
Keywords: base excision repair; trinucleotide repeat expansion; CAG triplets; Huntington’s disease; 8-oxoguanine; OGG1
Fluorescence studies on the interaction of myoglobin with mitochondria by G. B. Postnikova; E. A. Shekhovtsova (280-287).
To determine the nature and characteristic parameters of the myoglobin-mitochondrion interaction during oxymyoglobin (MbO2) deoxygenation in the cell, we studied the quenching of the intrinsic mitochondrial flavin and tryptophan fluorescence by different liganded myoglobins in the pH range of 6–8, as well as the quenching of the fluorescence of the membrane probes 1,8-ANS and merocyanine 540 (M 540) embedded into the mitochondrial membrane. Physiologically active MbO2 and oxidized metmyoglobin (metMb), which are unable to bind oxygen, were used as the quenchers. The absence of quenching of flavin and tryptophan fluorescence implies that myoglobin does not form quenching complexes with either electron transport chain proteins of the inner mitochondrial membrane or with outer membrane proteins. We found, however, that MbO2 and metMb effectively quench 1,8-ANS and M 540 fluorescence in the pH range of 6–8. Characteristic parameters of 1,8-ANS and M 540 fluorescence quenching by the myoglobins (extent of quenching and quencher binding constant, K m) are very similar, indicating that both probes are localized in phospholipid sites of the mitochondrial membrane, and myoglobin is complexed with these sites. The dependence of K m on ionic strength proves the important role of coulombic interactions in the formation of the quenching complex. Since the overall charge of myoglobin is shown not to influence the K m values, the ionic strength dependence must be due to local electrostatic interactions in which polar groups of some part of the myoglobin molecule participate. The most likely candidates to interact with anionic groups of mitochondrial phospholipids are invariant lysine and arginine residues in the environment of the myoglobin heme cavity, which do not change their ionization state in the pH range investigated.
Keywords: myoglobin; mitochondria; membrane; fluorescence
Characterization of recombinant PPi-dependent 6-phosphofructokinases from Methylosinus trichosporium OB3b and Methylobacterium nodulans ORS 2060 by O. N. Rozova; V. N. Khmelenina; Y. A. Trotsenko (288-295).
The properties of the purified recombinant PPi-dependent 6-phosphofructokinases (PPi-PFKs) from the methanotroph Methylosinus trichosporium OB3b and rhizospheric phytosymbiont Methylobacterium nodulans ORS 2060 were determined. The dependence of activities of PPi-PFK-His6-tag from Ms. trichosporium OB3b (6 × 45 kDa) and PPi-PFK from Mb. nodulans ORS 2060 (4 × 43 kDa) on the concentrations of substrates of forward and reverse reactions conformed to Michaelis-Menten kinetics. Besides fructose-6-phosphate, the enzymes also phosphorylated sedoheptulose-7-phosphate. ADP or AMP (1 mM each) inhibited activity of the Ms. trichosporium PPi-PFK but did not affect the activity of the Mb. nodulans enzyme. Preference of PPi-PFKs to fructose-1,6-bisphosphate implied a predominant function of the enzymes in hexose phosphate synthesis in these bacteria. PPi-PFKs from the methylotrophs have low similarity of translated amino acid sequences (17% identity) and belong to different phylogenetic subgroups of type II 6-phosphofructokinases. The relationship of PPi-PFKs with microaerophilic character of Ms. trichosporium OB3b and adaptation of Mb. nodulans ORS 2060 to anaerobic phase of phytosymbiosis are discussed.
Keywords: pyrophosphate-dependent 6-phosphofructokinase; methylotrophic bacteria; sedoheptulose-1,7-bisphosphatase; glycolysis; gluconeogenesis
Computer modeling of transketolase-like protein, TKTL1, a marker of certain tumor tissues by A. O. Maslova; L. E. Meshalkina; G. A. Kochetov (296-299).
A computer model of the spatial structure of transketolase-like protein (TKTL1), a marker of certain tumor tissues, has been constructed using the known spatial structure of transketolase found in normal human tissues. The structure of the two proteins at all levels of their organization has also been compared. On the basis of the revealed differences in structures of these proteins, we assume it is unlikely that TKTL1 can be a thiamine diphosphate-dependent protein capable of catalyzing the transketolase reaction.
Keywords: human transketolase; thiamine diphosphate; transketolase-like protein (TKTL1); computer modeling
Oligopeptidase B from Serratia proteamaculans. III. Inhibition analysis. Specific interactions with metalloproteinase inhibitors by A. G. Mikhailova; R. F. Khairullin; G. Ya. Kolomijtseva; L. D. Rumsh (300-306).
Inhibition of the novel oligopeptidase B from Serratia proteamaculans (PSP) by basic pancreatic trypsin inhibitor, Zn2+ ions, and o- and m-phenanthroline was investigated. A pronounced effect of calcium ions on the interaction of PSP with inhibitors was demonstrated. Inversion voltamperometry and atomic absorption spectrometry revealed no zinc ions in the PSP molecule. Hydrophobic nature of the enzyme inhibition by o- and m-phenanthroline was established.
Keywords: oligopeptidase B; Serratia proteamaculans ; inhibition analysis; zinc-dependent enzymes; atomic absorption spectrometry; inversion voltamperometry
Binding of DNA methyltransferase M.Ecl18 to operator-promoter region decreases its methylating activity by D. V. Nikitin; M. L. Mokrishcheva; A. S. Solonin (307-311).
The type II bifunctional DNA methyltransferase (MTase) Ecl18 that is able to control transcription of its own gene was studied kinetically. Based on initial velocity dependences from S-adenosyl-L-methionine (AdoMet) and target DNA and substrate preincubation assays, it is proposed that the enzyme apparently works by a rapid equilibrium ordered bi-bi mechanism with DNA binding first. By measuring the enzyme activity depending on DNA and AdoMet at different fixed concentrations of the operator sequence oligonucleotide, it was found that its binding has noncompetitive inhibitory effect on Ecl18 MTase activity.
Keywords: type II DNA methyltransferase; enzyme kinetics; noncompetitive inhibition