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Mitochondrial dysfunction and ubiquitin-proteasome system (UPS) failure contribute significantly to the development of Parkinson's disease (PD). The proteasome subunit Rpn13 located on the regulatory (19S) subparticle play an important role in the delivery of proteins, subjected to degradation, to the proteolytic (20S) part of proteasome. We have previously found several brain mitochondrial proteins specifically bound to Rpn13 (Buneeva et al. (2020) Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry, 14, 297-305). In this study we have investigated the effect of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the neuroprotector isatin on the mitochondrial subproteome of Rpn13-binding proteins of the mouse brain. Administration of MPTP (30 mg/kg) to animals caused movement disorders typical of PD, while pretreatment with isatin (100 mg/kg, 30 min before MPTP) reduced their severity. At the same time, the injection of MPTP, isatin, or their combination (isatin + MPTP) had a significant impact on the total number and the composition of Rpn13-binding proteins. The injection of MPTP decreased the total number of Rpn13-binding proteins in comparison with control, and the injection of isatin prior to MPTP or without MPTP caused an essential increase in the number of Rpn13-binding proteins, mainly of the functional group of proteins participating in the protein metabolism regulation, gene expression, and differentiation. Selected biosensor validation confirmed the interaction of Rpn13 subunit of proteasome with some proteins (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, histones H2A and H2B) revealed while proteomic profiling. The results obtained testify that under the conditions of experimental MPTP-induced parkinsonism the neuroprotective effect of isatin may be aimed at the interaction of mitochondria with the components of UPS.Mitokhondrial'naia disfunktsiia i sboĭ v rabote ubikvitin-proteasomnoĭ sistemy (UPS) vnosiat sushchestvennyĭ vklad v razvitie bolezni Parkinsona (BP). Sub"edinitsa proteasomy Rpn13, raspolozhennaia na reguliatornoĭ subchastitse (19S), igraet vazhnuiu rol' v dostavke belkov, podlezhashchikh degradatsii, v proteoliticheskuiu chast' (20S) proteasomy. Ranee nami obnaruzhen riad mitokhondrial'nykh belkov mozga, spetsificheski sviazyvaiushchikhsia s Rpn13 (Buneeva i dr., Biomeditsinskaia khimiia, 2020, 66(2), 138-144). V dannoĭ rabote izucheno vliianie neĭrotoksina 1-metil-4-fenil-1,2,3,6-tetragidropiridina (MFTP) i neĭroprotektora izatina na mitokhondrial'nyĭ subproteom Rpn13-sviazyvaiushchikh belkov mozga myshi. Vvedenie MFTP (30 mg/kg) zhivotnym vyzyvalo razvitie dvigatel'nykh narusheniĭ, svoĭstvennykh dlia BP, a predvaritel'noe vvedenie izatina (100 mg/kg) oslablialo ikh vyrazhennost'. Pri étom vvedenie MFTP, izatina ili ikh kombinatsii (izatin + MFTP) privodilo k izmeneniiu obshchego chisla i sostava Rpn13-sviazyvaiushchikh belkov. Vvedenie MFTP snizhalo obshchee chislo Rpn13-sviazyvaiushchikh belkov po sravneniiu s kontrolem, a vvedenie izatina pered vvedeniem MFTP ili bez MFTP privodilo k sushchestvennomu uvelicheniiu chisla Rpn13-sviazyvaiushchikh belkov preimushchestvenno iz funktsional'noĭ gruppy belkov, uchastvuiushchikh v reguliatsii metabolizma belkov, a takzhe ékspressii genov, kletochnogo deleniia i differentsirovki. Vyborochnaia biosensornaia validatsiia podtverdila vzaimodeĭstvie riada belkov (glitseral'degid-3-fosfatdegidrogenaza, piruvatkinaza, gistony N2A i N2V), vyiavlennykh v khode proteomnogo profilirovaniia, s sub"edinitseĭ proteasomy Rpn13. Poluchennye rezul'taty svidetel'stvuiut o tom, chto v usloviiakh éksperimental'nogo MFTP-indutsirovannogo parkinsonizma neĭroprotektornoe deĭstvie izatina mozhet byt' napravleno na vzaimodeĭstvie mitokhondriĭ s komponentami UPS.

In this article, an original method is proposed for address and covalent immobilization of anti-E. coli antibodies on a screen-printed electrode (SPE). The method is based on a copper-catalyzed “click” reaction between a polyvinylbenzylazide (PVBA) film electrochemically deposited on the electrode surface and acetylene fragments of propargyl-N-hydroxysuccinimide ester. The products of electrochemical oxidation of copper particles incorporated in the polymer film on the electrode were first used for catalysis of the click reaction. This approach allowed us to reduce the immobilization time from a few hours for conventional methods to just 30 min, and to prevent denaturation of the immunoreceptor. The modified electrodes were characterized by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). Based on the results obtained, a label-free impedimetric immunosensor for E. coli detection was developed. The detection limit of the immunosensor was estimated as 6.3 CFU/ml, with a linear range of 103–106 CFU/ml. The immunosensor demonstrated good stability during 30 days of storage in phosphate buffer solution (PBS, pH 7) and selectivity toward excess Staphylococcus aureus bacteria.

Background Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the major targets of NO in cells, especially in neurodegenerative diseases. S-Nitrosylation of GAPDH is accompanied by its translocation into the nucleus with subsequent apoptosis. The product of GAPDH modification by NO is considered to be S-nitrosylated GAPDH (GAPDH-SNO). However, this has not been confirmed by direct methods. Methods Products of GAPDH modification in the presence of the NO donor diethylamine NONOate were analyzed by MALDI- and ESI- mass spectrometry methods. Results The adduct between GAPDH and dimedone was detected by MALDI-MS analysis after incubation of S-nitrosylated GAPDH with dimedone, which points to the formation of cysteine-sulfenic acid (GAPDH-SOH) in the protein. Analysis of the protein hydrolysate revealed the incorporation of dimedone into the catalytic residue Cys150. An additional peak that corresponded to GAPDH-SNO was detected by ESI-MS analysis in GAPDH after the incubation with the NO donor. The content of GAPDH-SNO and GAPDH-SOH in the modified GAPDH was evaluated by different approaches and constituted 2.3 and 0.7 mol per mol GAPDH, respectively. A small fraction of GAPDH was irreversibly inactivated after NO treatment, suggesting that a minor part of the products includes cysteine-sulfinic or cysteine-sulfonic acids. Conclusions The main products of GAPDH modification by NO are GAPDH-SNO and GAPDH-SOH that is presumably formed due to the hydrolysis of GAPDH-SNO. General significance The obtained results are important for understanding the molecular mechanism of redox regulation of cell functions and the role of GAPDH in the development of neurodegenerative disorders.

Mitochondria play an important role in molecular mechanisms of neuroplasticity, adaptive changes of the brain that occur in the structure and function of its cells in response to altered physiological conditions or development of pathological disorders. Mitochondria are a crucial target for actions of neurotoxins, causing symptoms of Parkinson's disease in various experimental animal models, and also neuroprotectors. Good evidence exists in the literature that mitochondrial dysfunction induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) influences functioning of the ubiquitin-proteasomal system (UPS) responsible for selective proteolytic degradation of proteins from various intracellular compartments (including mitochondria), and neuroprotective effects of certain antiparkinsonian agents (monoamine oxidase inhibitors) may be associated with their effects on UPS. The 19S proteasomal Rpn10 subunit is considered as a ubiquitin receptor responsible for delivery of ubiquitinated proteins to the proteasome proteolytic machinery. In this study, we investigated proteomic profiles of mouse brain mitochondrial Rpn10-binding proteins, brain monoamine oxidase B (MAO B) activity, and their changes induced by a single-dose administration of the neurotoxin MPTP and the neuroprotector isatin. Administration of isatin to mice prevented MPTP-induced inactivation of MAO B and influenced the profile of brain mitochondrial Rpn10-binding proteins, in which two pools of proteins were clearly recognized. The constitutive pool was insensitive to neurotoxic/neuroprotective treatments, while the variable pool was specifically influenced by MPTP and the neuroprotector isatin. Taking into consideration that the neuroprotective dose of isatin used in this study can result in brain isatin concentrations that are proapoptotic for cells in vitro, the altered repertoire of mitochondrial Rpn10-binding proteins may thus represent a part of a switch mechanism from targeted elimination of individual (damaged) proteins to more efficient ("global") elimination of damaged organelles and whole damaged cells.

Amyloid-β peptide (1-42) (Aβ1-42) is a key player in the development and progression of Alzheimer's disease (AD) and related pathologies, determined by formation of protein aggregates in the central nervous system. Aβ1-42 binding to crucial intracellular targets (and their subsequent inactivation) obviously represents one of the earliest events preceding extracellular pathogenic oligomerization/aggregation of Aβ1-42. It is reasonable to expect that dissociation of the Aβ1-42 complexes with intracellular proteins by means of inhibitors followed by subsequent degradation of Aβ1-42 would not only protect critically important proteins but also prevent intracellular accumulation of Aβ1-42. The aim of this study was to investigate the effect of the neuroprotector isatin (100 mM) on interaction of known Aβ-binding proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase, with Aβ1-42 and its fragments (Aβ1-28, Aβ12-28, Aβ25-35). Aβ1-42 and its fragments (Aβ1-28, Aβ12-28, Aβ25-35) immobilized on the Biacore optical biosensor chip interacted with GAPDH and pyruvate kinase. The lowest and basically equal Kd values were determined for GAPDH and pyruvate kinase complexes with immobilized Aβ1-42 and Aβ25-35. The presence of 100 mM isatin caused a significant (more than fivefold) increase in the Kd values for GAPDH complexes with all Aβ peptides except Aβ1-28. In contrast to GAPDH isatin increased dissociation of pyruvate kinase complexes only with Aβ1-42 (causing a 30-fold increase in Kd) and to a lesser extent with Aβ12-28 and Aβ25-35 (a 10-fold increase in Kd). It should be noted that in the presence of isatin the Kd values for GAPDH and pyruvate kinase complexes with all Aβ studied were in a narrower concentration range (10-7 M - 10-6 M) than in the absence of this neuroprotector (10-8 M - 10-6 M). Data obtained suggest existence of principal possibility of (pharmacological) protection of crucial intracellular targets against both Aβ1-42, and its aggressive truncated peptides (Aβ25-35).Beta-amiloidnyĭ peptid (1-42) (Aβ1-42) iavliaetsia odnim iz “kliuchevykh igrokov”, vovlechennykh v razvitie bolezni Al'tsgeĭmera (AD) i drugikh patologicheskikh sostoianiĭ, obuslovlennykh obrazovaniem i nakopleniem belkovykh agregatov v tsentral'noĭ nervnoĭ sisteme. Ego sviazyvanie s vazhnymi vnutrikletochnymi misheniami s posleduiushcheĭ ikh inaktivatsieĭ, po-vidimomu, mozhet predstavliat' soboĭ odno iz rannikh sobytiĭ, predshestvuiushchikh formirovaniiu vnekletochnoĭ patogennoĭ oligomerizatsii/agregatsii Aβ1-42. Mozhno ozhidat', chto dissotsiatsiia kompleksov Aβ1-42 s belkami-misheniami pod deĭstviem ingibitorov kompleksoobrazovaniia i posleduiushchaia degradatsiia Aβ1-42 budet ne tol'ko zashchishchat' kriticheski vazhnye vnutrikletochnye misheni, no takzhe prepiatstvovat' nakopleniiu Aβ1-42 vnutri kletok. Tsel'iu dannoĭ raboty bylo issledovanie vliianiia neĭroprotektora izatina (100 mkM) na vzaimodeĭstvie izvestnykh Ab-sviazyvaiushchikh belkov – glitseral'degid-3-fosfatdegidrogenazy (GAFD) i piruvatkinazy – s Aβ1-42 i ego fragmentami (Aβ1-28, Aβ12-28, Aβ25-35). Immobilizovannye na chipe opticheskogo biosensora Biacore Aβ1-42 i ego fragmenty (Aβ1-28, Aβ12-28, Aβ25-35) vzaimodeĭstvovali s GAFD i piruvatkinazoĭ. Naibolee nizkie i primerno ravnye znacheniia Kd byli polucheny dlia kompleksov étikh fermentov s Aβ1-42 i Aβ25-35. V prisutstvii 100 mkM izatina sushchestvennoe uvelichenie Kd v 5 i bolee raz otmecheno dlia kompleksov GAFD so vsemi issledovannymi Ab peptidami za iskliucheniem Aβ1-28. V otlichie ot GAFD, izatin sposobstvoval dissotsiatsii kompleksov piruvatkinazy tol'ko s polnorazmernym Aβ1-42 (vyzyvaia uvelichenie Kd bolee chem v tridtsat' raz) i v men'sheĭ stepeni s Aβ25-35 i Aβ1-28 (uvelichenie Kd primerno na odin poriadok). Pri étom znacheniia Kd dlia kompleksov piruvatkinazy i GAFD so vsemi issledovannymi Ab peptidami v prisutstvii 100 mkM izatina nakhodilis' v bolee uzkom diapazone (10-7 M – 10-6 M), chem v otsutstvie étogo neĭroprotektora (10-8 M – 10-6 M). Poluchennye dannye svidetel'stvuiut o sushchestvovanii printsipial'noĭ vozmozhnosti (farmakologicheskoĭ) zashchity vazhnykh vnutrikletochnykh misheneĭ ot deĭstviia kak Aβ1-42, tak i ego agressivnykh ukorochennykh peptidov (Aβ25-35).

A series of novel D–A and D–π–A push-pull systems based on a pyrazine and quinoxaline acceptor, bearing various electron-donating triphenylamine and carbazole moieties, are compared. A significant difference in electrochemical and photophysical properties was found depending on molecular structure. The compounds have strong solvatochromic properties. Quinoxaline-containing systems exhibit delayed fluorescence (DF) in thermal vacuum deposition films. Despite the low quantum yield of fluorescence in the solid state (less than 10%), organic light-emitting diodes with sufficiently high efficiency (4.2 cd/A) have been fabricated on the basis of this push-pull systems. The best results were obtained for compounds exhibiting DF. The possible channel for increasing the efficiency of OLED can be associated with the “hot excitons” mechanism.

The amyloid-beta peptide 1-42 formed during proteolytic processing of the amyloid precursor protein (APP) plays a key role in the development or progression of Alzheimer's disease (AD) and other pathologies associated with formation of protein aggregates in the central nervous system. Recent proteomic profiling of mouse and rat brain preparations by means of beta-amyloid peptide immobilized on Affigel-10 revealed a large group of amyloid-binding proteins (n80). Many (about 25%) of these proteins were previously identified as isatin-binding proteins. The aim of this study was to validate direct interaction between beta-amyloid peptide and highly purified intact and oxidized peroxiredoxin, M-type pyruvate kinase, alpha-enolase, and the effect of isatin on this interaction. The study performed using SPR-based Biacore 3000 and Biacore X100 biosensors has shown that all the proteins form molecular complexes with immobilized beta-amyloid peptide. The Kd values for these complexes varied from 8.36х10-8 M (peroxiredoxin) to 1.97х10-6 M (alpha-enolase). Oxidative modification of investigated proteins caused opposite effects on complexes of these peptides with beta-amyloid. The endogenous neuroprotector isatin increased dissociation of complexes formed by beta-amyloid peptide with both intact proteins (peroxiredoxin, glyceraldehyde-3-phosphate dehydrogenase) and/or oxidized proteins (peroxiredoxin, pyruvate kinase) used in this study.Beta-amiloidnyĭ peptid (1-42), obrazuiushchiĭsia v rezul'tate proteoliticheskogo protsessinga transmembrannogo belka-predshestvennika amiloida, rassmatrivaetsia v kachestve "kliuchevogo igroka" v razvitii bolezni Al'tsgeĭmera (AD) i drugikh patologicheskikh sostoianiĭ, obuslovlennykh obrazovaniem i nakopleniem belkovykh agregatov v tsentral'noĭ nervnoĭ sisteme. V khode proteomnogo profilirovaniia preparatov mozga intaktnykh krys i mysheĭ, provedennogo ranee s ispol'zovaniem v kachestve affinnogo sorbenta affigelia-10 s immobilizovannym beta-amiloidnym peptidom, bylo identifitsirovano bolee 80 individual'nykh belkov, spetsificheski sviazyvaiushchikhsia s étim ligandom. Mnogie iz nikh byli obnaruzheny ranee kak belki, sviazyvaiushchiesia s éndogennym neĭroprotektorom izatinom. Tsel'iu dannogo issledovaniia byla priamaia éksperimental'naia proverka vzaimodeĭstviia vysokoochishchennykh intaktnykh i okislennykh preparatov peroksiredoksina, piruvatkinazy myshechnogo tipa i al'fa-enolazy s immobilizovannym beta-amiloidnym peptidom i vliianie izatina na étot protsess. Po dannym biosensornogo analiza s ispol'zovaniem opticheskikh SPR-biosensorov Biacore 3000 i Biacore X100 vse issledovannye belki obrazovyvali molekuliarnye kompleksy s immobilizovannym beta-amiloidnym peptidom. Znacheniia Kd étikh kompleksov var'irovali ot 8,36kh10-8 M (u peroksiredoksina) do 1,97kh10-6 M (u al'fa-enolazy). Okislitel'naia modifikatsiia issledovannykh belkov okazyvala raznonapravlennoe vliianie na prochnost' kompleksov s beta-amiloidnym peptidom, a éndogennyĭ neĭroprotektor izatin povyshal dissotsiatsiiu kompleksov beta-amiloidnogo peptida s riadom intaktnykh (peroksiredoksin, GAFD) i/ili podvergnutykh okislitel'noĭ modifikatsii (peroksiredoksin, piruvatkinaza) model'nykh belkov.

Anodic-corrosion behavior of a number of chromium-nickel steels in a 80% H2SO4 solution was studied in order to replace platinum with less expensive electrodes in electrochemical synthesis of graphite bisulfite and corrosion parameters were determined.

The glycolytic enzyme, pyruvate kinase, exhibits moderate affinity [3H]isatin binding (KD approximately 10 microM), which is inhibited by ATP (IC50 25 microM) and deprenyl (IC50 5 microM). Interaction of pyruvate kinase with isatin and its inhibition by ATP and deprenyl has also been confirmed using an independent biosensor technique and immobilized isatin analogue, aminoisatin. This effect has some specificity because the enzyme, creatine phosphokinase, does not exhibit specific isatin-binding. It is suggested that interaction of pyruvate kinase with isatin may reflect some non-glycolytic functions of this enzyme.

The amyloid-β peptide is considered as a key player in the development and progression of Alzheimer’s disease (AD). Although good evidence exists that amyloid-β accumulates inside cells, intracellular brain amyloid-binding proteins remain poorly characterized. Proteomic profiling of rat brain homogenates, performed in this study, resulted in identification of 89 individual intracellular amyloid-binding proteins, and approximately 25% of them were proteins that we had previously identified as specifically binding to isatin, an endogenous neuroprotector molecule. A significant proportion of the amyloid-binding proteins (more than 30%) are differentially expressed or altered/oxidatively modified in AD patients. Incubation of brain homogenates with 70 µM hydrogen peroxide significantly influenced the profile of amyloid-β binding proteins and 0.1 mM isatin decreased the number of identified amyloid-β binding proteins both in control and hydrogen peroxide treated brain homogenates. The effects of hydrogen peroxide and isatin have been confirmed in optical biosensor experiments with purified glyceraldehyde-3-phosphate dehydrogenase, one of the known crucial amyloid-β binding proteins (also identified in this study). Data obtained suggest that isatin protects crucial intracellular protein targets against amyloid binding, and possibly favors intracellular degradation of this protein via preventing formation of amyloid-β oligomers described in the literature for some isatin derivatives.

Interaction of five tryptic fragments of calmodulin with caldesmon and calponin was analysed by native gel electrophoresis. In the presence of Ca2+ intact calmodulin interacts with caldesmon and calponin with apparent Kd values equal to 0.23 and 1.3 microM respectively. The interaction was abolished in the absence of Ca2+. Two large tryptic fragments of calmodulin obtained in the presence of Ca2+ (TR1C, residues 1-77, and TR2C, residues 78-148) interact with caldesmon with apparent Kd values of 11.9 and 4.6 microM. Affinity of TR2C to calponin (Kd 3.8 microM) was comparable with that of native calmodulin and was much higher than the corresponding value for TR1C (Kd 41 microM). The short C-terminal tryptic peptide of calmodulin obtained in the presence of EGTA (TR3E, residues 107-148) interacts with caldesmon and calponin with Kd values of 23.9 and 12.1 microM, whereas the large N-terminal peptide TR1E (residues 1-106) interacts with both caldesmon and calponin with a very low affinity (Kd 60 microM). Thus although both N- and C-terminal domains of calmodulin are involved in the interaction with caldesmon and calponin, the C-terminal part of calmodulin (residues 78-148) is of special importance and has the highest contribution for caldesmon and calponin binding.

There is evidence that the binding of deprenyl, a monoamine oxidase (MAO) B inhibitor, and other propargylamines to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is primarily responsible for their neuroprotective and antiapoptotic effects. Thus, GAPDH may be a target for other neuroprotective drugs. Using two independent approaches, radioligand analysis and an optical biosensor technique, we demonstrate here that GAPDH also interacts with the endogenous, reversible MAO B inhibitor, isatin. Deprenyl inhibited both [3H]isatin binding to GAPDH, and the binding of this enzyme to an isatin analogue, 5-aminoisatin, immobilized on to an optical biosensor cell. Another MAO inhibitor, tranylcypromine, was ineffective. Both deprenyl and isatin inhibited GAPDH-mediated cleavage of E. coli tRNA, and their effects were not additive. We suggest that isatin may be an endogenous partial functional agonist of deprenyl in its effect on GAPDH and GAPDH-mediated RNA cleavage. Changes in level of endogenous isatin may influence the neuroprotective effect of deprenyl in vivo.