Biochemistry (Moscow) (v.82, #9)

On p. 973 the proper title of the article should read “How Fucose of Blood Group Glycotopes Programs Human Gut Microbiota” instead of “How Fucose of Blood Group Glycotypes Programs Human Gut Microbiota”.Formation of appropriate gut microbiota is essential for human health. The first two years of life is the critical period for this process. Selection of mutualistic microorganisms of the intestinal microbiota is controlled by the FUT2 and FUT3 genes that encode fucosyltransferases, enzymes responsible for the synthesis of fucosylated glycan structures of mucins and milk oligosaccharides. In this review, the mechanisms of the selection and maintenance of intestinal microorganisms that involve fucosylated oligosaccharides of breast milk and mucins of the newborn’s intestine are described. Possible reasons for the use of fucose, and not sialic acid, as the major biological signal for the selection are also discussed.
Keywords: intestinal microbiota; FUT2 and FUT3 genes; fucosyltransferases; fucose; sialic acids; plant polysaccharides; glycotopes

Klotho protein: Its role in aging and central nervous system pathology by I. S. Boksha; T. A. Prokhorova; O. K. Savushkina; E. B. Tereshkina (990-1005).
This review is devoted to Klotho protein and recent evidences for its functions in the brain. Information on transcriptional regulation of the klotho gene and posttranslational modifications of the protein resulting in multiple forms of Klotho is reviewed. Evidence is summarized that Klotho regulates the activity of protein factors, enzymes, and receptors, including data suggesting the importance of its glycosidase activity. Effects of Klotho on components of the glutamatergic neurotransmitter system, signal cascades involving protein kinases and protein phosphorylation, as well as oligodendrocyte differentiation and myelination are discussed. A possible contribution is proposed for Klotho levels in the development of central nervous system pathologies including mental disorders.
Keywords: Klotho protein; aging; brain; signal cascades; myelination; glutamate system; mental pathology

Effect of anesthetics on efficiency of remote ischemic preconditioning by D. N. Silachev; E. A. Usatikova; I. B. Pevzner; L. D. Zorova; V. A. Babenko; M. V. Gulyaev; Yu. A. Pirogov; E. Yu. Plotnikov; D. B. Zorov (1006-1016).
Remote ischemic preconditioning of hind limbs (RIPC) is an effective method for preventing brain injury resulting from ischemia. However, in numerous studies RIPC has been used on the background of administered anesthetics, which also could exhibit neuroprotective properties. Therefore, investigation of the signaling pathways triggered by RIPC and the effect of anesthetics is important. In this study, we explored the effect of anesthetics (chloral hydrate and Zoletil) on the ability of RIPC to protect the brain from injury caused by ischemia and reperfusion. We found that RIPC without anesthesia resulted in statistically significant decrease in neurological deficit 24 h after ischemia, but did not affect the volume of brain injury. Administration of chloral hydrate or Zoletil one day prior to brain ischemia produced a preconditioning effect by their own, decreasing the degree of neurological deficit and lowering the volume of infarct with the use of Zoletil. The protective effects observed after RIPC with chloral hydrate or Zoletil were similar to those observed when only the respective anesthetic was used. RIPC was accompanied by significant increase in the level of brain proteins associated with the induction of ischemic tolerance such as pGSK-3β, BDNF, and HSP70. However, Zoletil did not affect the level of these proteins 24 h after injection, and chloral hydrate caused increase of only pGSK-3β. We conclude that RIPC, chloral hydrate, and Zoletil produce a significant neuroprotective effect, but the simultaneous use of anesthetics with RIPC does not enhance the degree of neuroprotection.
Keywords: ischemia; brain; remote preconditioning; hind limb; chloral hydrate; Zoletil

In MCF-7 human breast carcinoma cells, α5β1 integrin hyperexpression, which was accomplished by transduction of a full-length α5 integrin cDNA, increased by about 50-70% the number of cells, survived during 48-72 h cell treatment with doxorubicin. Up-regulation of α5β1 reduced the level of the apoptogenic p53 protein and p21 cell cycle inhibitor, but enhanced the activity of Akt and mTOR protein kinases. In addition to these findings, we observed a significant decrease in the activity of both isoforms of phosphokinase Erk1/2, which is known to play a key role in cell viability pathways, including pathways alleviating stress damages caused by distinct antitumor drugs. Diminished Erk activity accompanying the rise of drug resistance can be explained by an “atypical” function of this kinase, which, in the cells studied, promotes an enhanced rather than reduced sensitivity to doxorubicin. To verify this suggestion, the effect of a specific Erk inhibitor, PD98059, on the resistance to doxorubicin of control and α5 cDNA-transduced MCF-7 cells was investigated. The data showed that suppression of Erk activity increased the resistance of control cells (transduced with an “empty” vector) to a level higher than that demonstrated by the α5 cDNA-transduced cells. The highest level of resistance was observed in α5β1trancduced cells treated with PD98059. Akt and mTOR kinase inhibitors had little if any effect on doxorubicin resistance of α5 cDNA-transduced MCF-7 cells. The data show for the first time that integrin α5β1 can stimulate drug resistance of tumor cells through a mechanism based on the inhibition of protein kinase Erk. From a more general view, the results of this investigation suggest that signal protein kinases can perform in tumor cells “non-canonical” functions, opposite to those, which are the basis for using kinase inhibitors in targeted cancer therapy. It follows that if a protein kinase is supposed to be used as a target for such therapy, it is important to explore its features in the particular tumor prior to the onset of treatment.
Keywords: integrins; tumor growth; drug resistance; Erk protein kinase; signaling

Changes in expression levels of genes encoding carbonic anhydrases α-CA1, α-CA2, α-CA4, β-CA1, β-CA2, βCA3, β-CA4, β-CA5, and β-CA6 in Arabidopsis thaliana leaves after light increase from 80 to 400 μmol PAR quanta·m−2·s−1 were investigated under short day (8 h) and long day (16 h) photoperiods. The expression of two forms of the gene, At3g01500.2 and At3g01500.3, encoding the most abundant carbonic anhydrase of leaves, β-CA1, situated in chloroplast stroma, was found. The content of At3g01500.3 transcripts was higher by approximately an order of magnitude compared to the content of At3g01500.2 transcripts. When plants were adapted to high light intensity under short day photoperiod, the expression level of both forms increased, whereas under long day photoperiod, the content of At3g01500.3 transcripts increased, and the content of transcripts of At3g01500.2 decreased. The expression levels of the At3g01500.3 gene and of genes encoding chloroplast carbonic anhydrases α-CA1, α-CA4, α-CA2 and cytoplasmic carbonic anhydrase β-CA2 increased significantly in response to increase in light intensity under short day, and these of the first three genes increased under long day as well. The expression level of the gene encoding α-CA2 under long day photoperiod as well as of genes of chloroplast β-CA5 and β-CA4 from plasma membranes and mitochondrial β-CA6 under both photoperiods depended insignificantly on light intensity. Hypotheses about the roles in higher plant metabolism of the studied carbonic anhydrases are discussed considering the effects of light intensity on expression levels of the correspondent genes.
Keywords: photosynthesis; Arabidopsis ; light intensity; photoperiod; gene expression; carbonic anhydrase; chloroplasts

Interaction of cholera toxin B-subunit with human T-lymphocytes by E. V. Navolotskaya; V. B. Sadovnikov; D. V. Zinchenko; Y. A. Zolotarev; V. M. Lipkin; V. P. Zav’yalov (1036-1041).
In this work, 125I-labeled cholera toxin B-subunit (CT-B) (specific activity 98 Ci/mmol) was prepared, and its high-affinity binding to human blood T-lymphocytes (K d = 3.3 nM) was determined. The binding of the 125I-labeled CT-B was inhibited by unlabeled interferon-α2 (IFN-α2), thymosin-α1 (TM-α1), and by the synthetic peptide LKEKK, which corresponds to sequences 16-20 of human TM-α1 and 131-135 of IFN-α2 (K i 0.8, 1.2, and 1.6 nM, respectively), but was not inhibited by the unlabeled synthetic peptide KKEKL with inverted sequence (K i > 1 μM). In the concentration range of 10-1000 nM, both CT-B and peptide LKEKK dose-dependently increased the activity of soluble guanylate cyclase (sGC) but did not affect the activity of membrane-bound guanylate cyclase. The KKEKL peptide tested in parallel did not affect sGC activity. Thus, the CT-B and peptide LKEKK binding to a common receptor on the surface of T-lymphocytes leads to an increase in sGC activity.
Keywords: peptides; receptors; thymosin-α1 ; interferon-α; T-lymphocytes

Quantitative affinity interaction of ubiquitinated and non-ubiquitinated proteins with proteasome subunit Rpn10 by O. A. Buneeva; O. V. Gnedenko; A. T. Kopylov; M. V. Medvedeva; V. G. Zgoda; A. S. Ivanov; A. E. Medvedev (1042-1047).
Recent proteomic profiling of mouse brain preparations using the ubiquitin receptor, Rpn10 proteasome subunit, as an affinity ligand revealed a representative group of proteins bound to this sorbent (Medvedev, A. E., et al. (2017) Biochemistry (Moscow), 82, 330-339). In the present study, we investigated interaction of the Rpn10 subunit of proteasomes with some of these identified proteins: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase, and histones H2A and H2B. The study revealed: (i) quantitative affinity interaction of the proteasome subunit immobilized on a Biacore-3000 optical biosensor cuvette with both the GAPDH (K d = 2.4·10–6 M) and pyruvate kinase (K d = 2.8·10–5 M); (ii) quantitative high-affinity interaction of immobilized histones H2A and H2B with the Rpn10 subunit (Kd values of 6.5·10–8 and 3.2·10–9 M, respectively). Mass spectrometric analysis revealed the presence of the ubiquitin signature (GG) only in a highly purified preparation of GAPDH. We suggest that binding (especially high-affinity binding) of non-ubiquitinated proteins to the Rpn10 proteasome subunit can both regulate the functioning of this proteasomal ubiquitin receptor (by competing with ubiquitinated substrates) and promote activation of other pathways for proteolytic degradation of proteins destined to the proteasome.
Keywords: Rpn10 proteasome subunit; Rpn10-binding proteins; optical biosensor; ubiquitin signature

Human lung cancer cells (Calu-3 line) were studied for the development of apoptosis, necrosis, and autophagy in response to infection with orthoand paramyxoviruses. Biochemical pathways underlying various mechanisms of cell death differed for different viruses. When infected with murine Sendai paramyxovirus, Calu-3 cells demonstrated typical necrotic features such as cell swelling (but not shrinkage), lack of chromatin DNA laddering, of caspase 3 and 8 activation, and of apoptotic cleavage of poly(ADP-ribose) polymerase (PARP) protein; an activation of antiapoptotic protein kinase Akt was also revealed. In contrast, infection with avian influenza virus A/FPV/Rostock/34 (H7N1 subtype) or Newcastle disease virus (NDV, avian paramyxovirus) caused the development of typical apoptotic markers such as cell shrinkage, ladder-type chromosomal DNA fragmentation, caspase 3 and 8 activation, and proteolytic cleavage of PARP in the absence of Akt activation. Notably, no upregulation of p53 protein phosphorylation was observed in all infected cells, which indicates that p53 is not involved in the virus-induced death of Calu-3 cells. Cell death caused by the influenza virus was accompanied by overstimulation of autophagy, whereas no stimulation of autophagy was observed in the NDV-infected cells. Infection with Sendai virus caused moderate stimulation of autophagy, which suggests that the mechanism of the virus-induced cell death and the balance between autophagy and cell death in infected cancer cells depend on the virus type and might significantly differ even for closely related viruses. Therefore, an optimal strategy for oncolytic virus-mediated destruction of tumor cells in cancer patients requires selection of the most appropriate oncolytic virus based on the mechanism of its cytolytic action in a particular type of tumor.
Keywords: Sendai virus; Newcastle disease virus; influenza virus; apoptosis; necrosis; autophagy; oncolytic viruses

The essential amino acid threonine is not synthesized in vertebrates, so it must be obtained from food. During evolution, the decomposition of threonine has changed. Because the decomposition of threonine catalyzed by threonine dehydratase is irreversible, in the present work attention is focused on threonine dehydrogenase to show the inability of this enzyme to synthesize threonine in a reaction that would be the reverse of the reaction of threonine decomposition. The reason why threonine dehydrogenase cannot be used for the biosynthesis of threonine in mammalian tissues is discussed. It is concluded that some quantity of threonine is involved in transamination.
Keywords: threonine; NAD; acetyl-CoA

The role of phosphatidylinositol-3 kinase (PI3K) and protein kinase A (PKA) in leptin and ghrelin regulation of formation of adaptive (a) subpopulations of CD4+ T-lymphocytes (helper (h) cells producing interleukin-17A) (aTh17) and of T-regulatory lymphocytes (aTreg) in the context of physiological pregnancy is established. It is shown that leptin at a concentration typical for the second half of pregnancy (trimesters II-III) enhances the differentiation of aTh17 with a high level of interleukin-17A (IL-17A) production and the expression of the chemokine receptor CCR6 with the participation of PI3K. Simultaneously, leptin reduces formation of aTreg expressing the suppressor molecule CTLA-4, which determines the function of these cells. Ghrelin at a concentration characteristic of the first half of pregnancy (trimesters I-II), in contrast, enhances aTreg formation and, in parallel, reduces the level aTh17 (that express CCR6) and the IL-17A production by aTh17. PKA, likewise PI3K, participates in regulatory effects of ghrelin on the formation of aTh17 and aTreg. The combined action of leptin and ghrelin (via PKA participation) enhances formation of only aTreg, which determines the priority of this molecular mechanism and explains why the investigated hormones with reciprocal differentiating potential do not come into antagonism at the level of immune system cells during pregnancy.
Keywords: leptin; ghrelin; aTh17; aTreg; IL-17A; PKA; PI3K

Comparison of interaction between ceruloplasmin and lactoferrin/transferrin: to bind or not to bind by A. V. Sokolov; I. V. Voynova; V. A. Kostevich; A. Yu. Vlasenko; E. T. Zakharova; V. B. Vasilyev (1073-1078).
The year 2016 marked the 50th anniversary of the discovery by S. Osaki who first showed that ceruloplasmin (CP, ferro:O2-oxidoreductase or ferroxidase) is capable of oxidizing Fe(II) to Fe(III) and favors the incorporation of the latter into transferrin (TF). However, much debate remains in the literature concerning the existence of a complex between the enzyme oxidizing iron and the protein facilitating its transport in plasma. We studied CP in exocrine fluids and demonstrated its high-affinity interaction with transferrin found in breast milk and in lacrimal fluid, i.e. with lactoferrin (LF). Here we present data obtained by comparing the interaction of CP with LF and TF using surface plasmon resonance and Hummel–Dreyer chromatography. Binding of apo-LF within the range of concentrations 1.6-51.3 μM with CP immobilized on a CM5-chip is characterized by K D = 1.07 μM. Under similar conditions, the K D for apo-TF was measured and appeared to be higher than 51.3 μM. Hummel–Dreyer chromatography of CP with 51 μM apo-LF/apo-TF in the effluent demonstrated the absence of interaction between apo-TF and CP in solution, contrary to efficient interaction between apoLF and CP. In contrast to LF, the interaction of apo-TF with CP is probably not stable within the physiological range of concentrations of TF.
Keywords: ceruloplasmin; transferrin; lactoferrin; protein–protein interaction; surface plasmon resonance; Hummel–Dreyer chromatography