Biochemistry (Moscow) (v.79, #8)

Cancer-retina antigens — A new group of tumor antigens by M. O. Golovastova; A. V. Bazhin; P. P. Philippov (733-739).
Some photoreceptor proteins normally specific for the eye retina are aberrantly expressed in malignant tumors. These proteins include recoverin, visual rhodopsin, transducin, cGMP-phosphodiesterase 6 (PDE 6), cGMP-dependent cationic channels, guanylyl cyclase 1, rhodopsin kinase, and arrestin. By analogy with cancer-testis antigens, these photoreceptor proteins form the group of cancer-retina antigens. It is shown that an aberrant demethylation of the promoter region of recoverin is involved in the aberrant expression of this protein. The cascade Wnt5a → Frizzled-2 → transducin → PDE 6 is shown to function in skin melanoma cells, and this suggests that these cancer-retina antigens can play a functional role. The events accompanying the signal transduction in this cascade, including those involving calcium ions and cGMP-dependent protein kinase (protein kinase G), are discussed.
Keywords: visual transduction; retina; cancer-retina antigens; aberrant expression; cancer

Lithium salts — Simple but magic by E. Y. Plotnikov; D. N. Silachev; L. D. Zorova; I. B. Pevzner; S. S. Jankauskas; S. D. Zorov; V. A. Babenko; M. V. Skulachev; D. B. Zorov (740-749).
For many decades pharmacological drugs based on lithium salts have been successfully used in psychiatry to treat bipolar disorder, and they remain the “gold standard” of pharmacological therapy of patients with this disease. At the same time, over recent years in experiments in vitro and in vivo a plethora of evidence has accumulated on a positive effect of lithium ions in other areas including their neuro-, cardio-, and nephroprotective properties, regulation of stem cells functions, regulation of inflammation, and others. Numerous studies have shown that the effect of lithium ions involves several mechanisms; however, one of its main targets in the implementation of most of the effects is glycogen synthase kinase 3β, a key enzyme in various pathological and protective signaling pathways in cells. However, one of the main limitations of the use of lithium salts in clinics is their narrow therapeutic window, and the risk of toxic side effects. This review presents the diversity of effects of lithium ions on the organism emphasizing their potential clinical applications with minimal undesirable side effects. In the end, we present a schematic “Lithiometer”, comparing the range of Li+ concentrations that might be used for the treatment of acute pathologies with possible toxic effects of Li+.
Keywords: lithium ions; glycogen synthase kinase; ischemia; brain; heart; kidney; mitochondria; stem cells

Mechanisms of sensing and adaptive responses to low oxygen conditions in mammals and yeasts by T. A. Trendeleva; D. A. Aliverdieva; R. A. Zvyagilskaya (750-760).
Oxygen is required for effective production of ATP and plays a key role in the maintenance of life for all organisms, excepting strict anaerobes. The ability of aerobic organisms to sense and respond to changes in oxygen level is a basic requirement for their survival. Eukaryotes have developed adaptive mechanisms to sense and respond to decreased oxygen concentrations (hypoxia) through adjustment of oxygen homeostasis by upregulating hypoxic and downregulating aerobic nuclear genes. This review summarizes recent data on mechanisms of cells sensing and responding to changes in oxygen availability in mammals and in yeasts. In the first part of the review, prominence is given to functional regulation and stabilization of hypoxia-inducible factors (HIFs), HIF-mediated regulation of electron transport flux and repression of lipogenesis, as well as to hypoxia-induced mitochondrial permeability transition (pore) opening, cell death, and autophagy. In the second part of the review emphasis is placed on oxygen sensing in nonpathogenic yeasts by heme, unsaturated fatty acids, and sterols, as well as on responses to hypoxia in fungal pathogens.
Keywords: hypoxia; yeasts; HIF; FIH; Hap1p; SREBP; mPTP

Prions and chaperones: Friends or foes? by Y. Y. Stroylova; G. G. Kiselev; E. V. Schmalhausen; V. I. Muronetz (761-775).
This review highlights the modern perception of anomalous folding of the prion protein and the role of chaperones therein. Special attention is paid to prion proteins from mammalian species, which are prone to amyloid-like prion diseases due to a unique aggregation pathway. Despite being a significantly popular current subject of investigations, the etiology, structure, and function of both normal and anomalous prion proteins still hold many mysteries. The most interesting of those are connected to the interaction with chaperone system, which is responsible for stabilizing protein structure and disrupting aggregates. In the case of prion proteins the following question is of the most importance — can chaperones influence different stages of the formation of pathological aggregates (these vary from intermediate oligomers to mature amyloid-like fibrils) and the whole transition from native prion protein to its amyloid-like fibril-enriched form? The existing inconsistencies and ambiguities in the observations made so far can be attributed to the fact that most of the investigations did not take into account the type and functional state of the chaperones. This review discusses in detail our previous works that have demonstrated fundamental differences between eukaryotic and prokaryotic chaperones in the action exerted on the amyloid-like transformation of the prion protein along with the dependence of the observed effects on the functional state of the chaperone.
Keywords: prion; chaperones; GroEL; TRiC (CCT); amyloid transformation of proteins; aggregation; neurodegenerative diseases

As we have shown previously, transcription of the rpsB-tsf operon encoding essential components of the translation machinery, a ribosomal protein S2 and an elongation factor Ts, is driven by a single promoter P rpsB , which is highly conserved among γ-proteobacteria. P rpsB belongs to the extended “-10” promoter class; it comprises a TGTG-extension upstream of the “-10” hexamer TATAAA, a suboptimal “-35” region TTGGTG, and a GC-rich discriminator GCGCGC that separates the “-10” element from the transcription start site. In this work, we examined an impact of site-directed mutations in the rpsB promoter region on expression of the reporter gene P rpsB -lacZ within the E. coli chromosome as well as promoter regulation by transcription factors ppGpp and DksA upon amino acid starvation. The results show that the transcription level largely depends on both the TGTG-extension and the TTG-element in the “−35” region, as mutations in these sequences dramatically decrease the activity of the promoter. Upon induction of amino acid starvation, the rpsB promoter is negatively regulated by ppGpp due to the presence of the GC-rich discriminator, whose substitution for the AT-rich element abolished stringent control. These and other data obtained demonstrate the necessity of a natural combination of all the conserved promoter elements for efficient and regulated transcription of the essential rpsB-tsf operon.
Keywords: rpsB-tsf operon; extended “-10” promoter; site-directed mutagenesis; ppGpp/DksA; GC-rich discriminator; stringent control

Papain, a cysteine protease isolated from the latex of Carica papaya, is known to undergo irreversible thermal unfolding. In this study, we found that thermal unfolding of papain is accompanied by a simultaneous self-assembly process where this protein is observed to aggregate above 50°C. The extent of aggregation increased with increasing protein concentration from 3–40 μM. The aggregation was confirmed by enhanced turbidity, light scattering intensity, 1-anilino-8-naphthalene sulfonate (ANS) fluorescence intensity and by transmission electron microscopy. Furthermore, we noted that post-micellar concentration of sodium dodecyl sulfate (SDS) remarkably suppresses the thermal aggregation of papain. Far-UV circular dichroism studies revealed that SDS significantly enhances α-helical content of the protein and also tends to prevent its unfolding, and thus inhibits aggregation. Additionally, papain showed maximal activity at 65°C in neutral buffer. However, in the presence of 6 mM SDS (above its critical micellar concentration), the enzyme lost activity by about 10-fold. Thus, promoting the helical propensity of the protein does not appear to be a suitable strategy to overcome the aggregation related problems of industrially important proteins such as papain, which are not only required to be protected against aggregation but also need to remain functionally active in the presence of aggregation inhibitors.
Keywords: papain; thermal aggregation; sodium dodecyl sulfate (SDS); critical micellar concentration (CMC); circular dichroism; 1-anilino-8-naphthalene sulfonate (ANS)

Immediately upon contact with blood, nanosized drug delivery systems become coated with a so-called protein corona. The quantitative and qualitative composition of the corona defines not only the behavior of the nanocarrier in the circulation but, ultimately, the pharmacokinetics and biodistribution of the encapsulated drug as well. In turn, the composition of the protein corona depends on the surface properties of the nanoparticles, such as size and distribution of charge and functional groups on the particle surface. Liposomes belong to the most bio- and hemocompatible drug delivery systems feasible for intravenous route of administration required in chemotherapy of metastasizing tumors. However, knowledge on the interactions of liposomes of various compositions with blood plasma proteins remains fragmentary. Moreover, all nanosized drug delivery systems are potential targets for the innate immunity system, primarily the complement (C) system, which underlies frequent cases of hypersensitivity reactions. Recently, in a panel of in vitro hemocompatibility tests, we demonstrated that liposomes built of natural phospholipids — egg phosphatidylcholine and phosphatidylinositol from Saccharomyces cerevisiae — and loaded with diglyceride conjugates of anticancer drugs melphalan and methotrexate, did not affect the morphology and numbers of the main blood cell types. While preparations with melphalan prodrug were also inert in coagulation and C activation tests, methotrexate-loaded liposomes caused impaired coagulation and C activation. The aim of this work was to study the interactions of liposomes carrying prodrugs of melphalan and methotrexate with blood plasma proteins in vitro. Data on protein binding capacity of liposomes obtained with classical gel permeation chromatography techniques allowed for prediction of rather rapid elimination of the liposomes from circulation. A number of differences revealed through immunoblotting of the liposome-bound proteins agree with the previously obtained data on C activation. The possible mechanism of C activation by methotrexate-containing liposomes is discussed.
Keywords: nanomedicine; antitumor liposomes; melphalan; methotrexate; lipophilic prodrugs; protein corona; complement

The effect of polypeptides having different charge on the activity of Thiocapsa roseopersicina HydSL hydrogenase was studied. Strong inhibition was shown for poly-L-lysine bearing positive charge. The inhibition was reversible and competitive to methyl viologen, an electron acceptor, in the reaction of hydrogen oxidation catalyzed by the hydrogenase. Peptides carrying less positive charge had weaker inhibiting effect, while neutral and negatively charged peptides did not inhibit the hydrogenase. Molecular docking of poly-L-lysine to T. roseopersicina hydrogenase showed strong affinity of this polypeptide to the acceptor-binding site of the enzyme. The calculated binding constant is close to the experimentally measured value (K i = 2.1 μM).
Keywords: hydrogenase; methyl viologen; polypeptides; competitive inhibition; molecular docking

The primary objective of this study was to determine the role of β2 integrin α-subunit (CD11b) in the mechanism of human polymorphonuclear leukocyte (PML) priming by S or Re endotoxin glycoforms from Escherichia coli for fMLP-induced respiratory burst. Similar priming activity of S and Re endotoxin glycoforms for fMLP-induced reactive oxygen species (ROS) generation from primed PML was found. Anti-CD11b antibodies (clone ICRF 44) as well as isotypematched immunoglobulin G1 (clone MOPC-21) do not influence the fMLP-induced ROS generation from unprimed PML. Antibodies against CD11b do not change fMLP-induced ROS generation from endotoxin-primed PML as well. The involvement of different isoforms of Fcγ receptors in fMLP-induced ROS generation from activated PML is proposed.
Keywords: lipopolysaccharide (endotoxin); β2 integrins (CD11b/CD18); Toll-like receptor 4; Fcγ receptors; receptor cluster; immunoglobulins; anti-human CD11b antibody; reactive oxygen species

11S storage globulin from pumpkin seeds: Regularities of proteolysis by papain by A. S. Rudakova; S. V. Rudakov; I. A. Kakhovskaya; A. D. Shutov (820-825).
Limited proteolysis of the α- and β-chains and deep cleavage of the αβ-subunits by the cooperative (one-by-one) mechanism was observed in the course of papain hydrolysis of cucurbitin, an 11S storage globulin from seeds of the pumpkin Cucurbita maxima. An independent analysis of the kinetics of the limited and cooperative proteolyses revealed that the reaction occurs in two successive steps. In the first step, limited proteolysis consisting of detachments of short terminal pep-tides from the α- and β-chains was observed. The cooperative proteolysis, which occurs as a pseudo-first order reaction, started at the second step. Therefore, the limited proteolysis at the first step plays a regulatory role, impacting the rate of deep degradation of cucurbitin molecules by the cooperative mechanism. Structural alterations of cucurbitin induced by limited proteolysis are suggested to generate its susceptibility to cooperative proteolysis. These alterations are tentatively discussed on the basis of the tertiary structure of the cucurbitin subunit pdb|2EVX in comparison with previously obtained data on features of degradation of soybean 11S globulin hydrolyzed by papain.
Keywords: Cucurbita maxima ; seed storage globulin; limited proteolysis; papain; kinetics

Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo by A. Y. Anikaev; A. P. Korepanov; A. V. Korobeinikova; V. G. Kljashtorny; W. Piendl; S. V. Nikonov; M. B. Garber; G. M. Gongadze (826-835).
5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.
Keywords: 5S rRNA-binding protein L25; RNA-protein interactions; ribosome; translation; Escherichia coli

Protease inhibitors (PIs) are generally small proteins that have been identified in plants. The wip1 gene codes for wound-induced protein, which is similar to serine PIs of the Bowman-Birk family (BBIs). In this study, we analyzed 10 wip1 genes of Turkish maize varieties to understand the structure and characteristics of the wip1 genes and proteins in maize. We found that genetic variability of wip1 genes was higher (π: 0.0173) than reported in previous studies. Tajima’s D value was found to be positive (1.73), suggesting over-dominant selection in these loci. According to phylogenetic analysis of wip1 proteins, monocot and dicot BBIs were separated independently, and Turkish varieties were clustered with each other generally. The 3D structures of wip1 proteins indicated that several wip1 proteins had structural divergence in active loops, containing various numbers of cysteine residues ranging between 7 and 9. Particularly, Cys74 was identified in Kocbey and Gozdem varieties, whereas Cys98 was only in the Gozdem variety. Also, a critical serine residue (Ser98) was observed in two varieties — Antbey and Batem Efe. These results can contribute to understanding the role of wip1 genes and corresponding proteins in maize.
Keywords: protease inhibitors; wound-induced protein; Bowman-Birk family; Turkish maize

A recent study published by Muslikhov et al. (Biochemistry (Moscow), 79, 435–439 (2014)) showed that arachidonic acid increases cytosolic Ca2+ concentrations in C2C12 skeletal myotubes mainly via activation of the ryanodine (RY) receptor 1. These results are consistent with the data from another study demonstrating that arachidonic acid targets RY receptor 2 in clonal and primary pancreatic β-cells (Woolcott et al., 2006). A novel and intriguing finding by Muslikhov’s group is that arachidonic acid also appears to activate the two-pore ion channel (TPC), suggesting that arachidonic acid could be a mediator in the interaction between TPCs and RY receptors.
Keywords: calcium ions; skeletal myotubes; arachidonic acid; ryanodine-sensitive channels