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It becomes increasingly clear that ubiquitination of cellular proteins is not an indispensable prerequisite of their degradation in proteasomes. There are a number of proteins to be eliminated which are not pre-ubiquitinated for their recognition by regulatory subcomplex of 26S proteasome, but which directly interact with the 20S proteasome core particle (20S proteasome). The obligatory precondition for such interaction consists in existence of disordered (hydrophobic) fragments in the target protein. In this study we have investigated the interaction of a number of multifunctional (moonlighting) proteins (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase) and neurodegeneration-related proteins (a-synuclein, myelin basic protein) with 20S proteasome immobilized on the SPR-biosensor chip and stabilized by means of a bifunctional agent dimethyl pimelimidate (in order to prevent possible dissociation of this subcomplex). Only two of all investigated proteins (aldolase and pyruvate kinase) interacted with the immobilized 20S proteasome (Kd of 8.17´10-7 M and 5.56´10-7 M, respectively). In addition to earlier detected GAPDH ubiquitination, mass spectrometric analysis of the studied proteins revealed the presence of the ubiquitin signature (Lys-e-Gly-Gly) only in aldolase. Oxidation of aldolase and pyruvate kinase, which promotes elimination of proteins via their direct interaction with 20S proteasome, caused a 2-3-fold decrease in their Kd values as comparison with this parameter obtained for the intact proteins. The results of this study provide further evidence for direct interaction of both ubiquitinated proteins (aldolase), and non-ubiquitinated proteins (pyruvate kinase) with the 20S proteasome core particle (20S proteasome). The effectiveness of this interaction is basically equal for the ubiquitinated proteins and non-ubiquitinated proteins.Stanovitsia vse bolee ochevidnym, chto ubikvitinirovanie vnutrikletochnykh belkov ne iavliaetsia obiazatel'nym étapom, neobkhodimym dlia ikh degradatsii v proteasomakh. Izvestno nemalo podlezhashchikh éliminatsii belkov, kotorye ne podvergaiutsia predvaritel'nomu ubikvitinirovaniiu dlia uznavaniia reguliatornymi subchastitsami 26S proteasomy, a postupaiut neposredstvenno v ee korovuiu (proteoliticheskuiu) chast' – 20S proteasomu. Neobkhodimym usloviem dlia étogo iavliaetsia nalichie v belke nestrukturirovannykh uchastkov. V dannoĭ rabote issledovali sviazyvanie riada polifunktsional'nykh belkov (glitseral'degid-3-fosfatdegidrogenaza (GAFD), al'dolaza, piruvatkinaza), a takzhe belkov, uchastvuiushchikh v protsessakh neĭrodegeneratsii (a-sinuklein, osnovnoĭ belok mielina), s immobilizovannoĭ na chipe SPR-biosensora Biacore 20S proteasomoĭ. Dlia predotvrashcheniia vozmozhnoĭ dissotsiatsii 20S proteasomu v khode immobilizatsii stabilizirovali pri pomoshchi bifunktsional'nogo sshivaiushchego reagenta – dimetilpimelimidata. Iz vsekh issledovannykh belkov tol'ko al'dolaza i piruvatkinaza vzaimodeĭstvovali s 20S proteasomoĭ (Kd sootvetstvenno 8,17´10-7 M i 5,56´10-7 M). V dopolnenie k ranee obnaruzhennomu ubikvitinirovaniiu GAFD, mass-spektrometricheskiĭ analiz preparatov étikh belkov vyiavil nalichie tak nazyvaemoĭ ubikvitinovoĭ signatury Lys-e-Gly-Gly tol'ko v preparate al'dolazy. Okislenie piruvatkinazy i al'dolazy, sposobstvuiushchee éliminatsii belkov v rezul'tate ikh neposredstvennogo vzaimodeĭstviia s 20S proteasomoĭ, privodilo k snizheniiu Kd v 2-3 raza po sravneniiu s intaktnymi belkami. Poluchennye rezul'taty svidetel'stvuiut v pol'zu togo, chto s 20S proteasomoĭ odinakovo éffektivno sviazyvaiutsia kak ubikvitinirovannye (al'dolaza), tak i neubikvitinirovannye (piruvatkinaza) belki.

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.