[The use of immobilized ubiquitin for biosensor analysis of the mitochondrial subinteractome]

Protein ubiquitination is considered as an important mechanism that is responsible not only for specific labeling of proteins for their subsequent degradation but also for localization of proteins in the cell and regulation of protein-protein interactions. In the context of protein-protein interactions binding of (mono/poly)ubiquitinated molecules to proteins containing specific ubiquitin binding domains appear to play the decisive role. Although formation of the ubiquitin interactome has been demonstrated for cytosol, involvement of mitochondria and associated extramitochondrial proteins into such interactions still requires detailed investigation. In this study using an optical biosensor we have demonstrated binding of proteins of mouse brain mitochondrial lysates to immobilized monomeric ubiquitin. Model purified proteins, which are known to be associated with the outer mitochondrial compartment (glyceraldehyde-3-phosphate dehydorgenase, creatine phosphokinase), interacted with immobilized ubiquitin as well as with each other. This suggests that (poly)ubiquitinated chains may be involved in protein-protein interactions between ubiquitinated and non-ubiquitinated proteins and thus may contribute to formation of (mitochondrial) ubiquitin subinteractome.Ubikvitinirovanie belkov iavliaetsia vazhnym mekhanizmom, kotoryĭ ne tol'ko obespechivaet spetsificheskoe mechenie dlia posleduiushcheĭ degradatsii, no i vo mnogom opredeliaet lokalizatsiiu belkov v kletke i reguliatsiiu belok-belkovykh vzaimodeĭstviĭ. V kontekste belok-belkovykh vzaimodeĭstviĭ reshaiushchuiu rol' igraet prisoedinenie (mono/poli)ubikvitinovykh molekul k belkam, soderzhashchim spetsificheskie ubikvitin-sviazyvaiushchie domeny. Formirovanie ubikvitinovogo interaktoma pokazano dlia tsitozolia. Vovlechenie v takogo roda vzaimodeĭstviia mitokhondriĭ i assotsiirovannykh s nimi ékstramitokhondrial'nykh belkov izucheno znachitel'no khuzhe. V nastoiashchem issledovanii s ispol'zovaniem opticheskogo biosensora pokazano sviazyvanie belkov lizata mitokhondrial'noĭ fraktsii mozga mysheĭ s immobilizovannym monomernym ubikvitinom. Model'nye ochishchennye belki (glitseral'degid-3-fosfatdegidrogenaza, kreatinkinaza), assotsiirovannye s vneshnim kompartmentom mitokhondriĭ, vzaimodeĭstvovali kak s immobilizovannym ubikvitinom, tak i mezhdu soboĭ. Éto svidetel'stvuet v pol'zu togo, chto (poli)ubikvitinovye tsepi mogut uchastvovat' v belok-belkovykh vzaimodeĭstviiakh mezhdu ubikvitinirovannymi i neubikvitinirovannymi belkami i vnosit' opredelennyĭ vklad v formirovanie (mitokhondrial'nogo) ubikvitinovogo subinteraktoma.