Buneeva Olga A, Medvedeva Marina V, Kopylov Arthur T, Zgoda Victor G, Medvedev Alexei E
Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, 10 Pogodinskaya street, Moscow 119121, Russia.
Moscow State University, Moscow, 119991, Russia.
Int J Mol Sci. 2012;13(9):11593-11609. doi: 10.3390/ijms130911593. Epub 2012 Sep 14.
Applicability of in vitro biotinylated ubiquitin for evaluation of endogenous ubiquitin conjugation and analysis of ubiquitin-associated protein-protein interactions has been investigated. Incubation of rat brain mitochondria with biotinylated ubiquitin followed by affinity chromatography on avidin-agarose, intensive washing, tryptic digestion of proteins bound to the affinity sorbent and their mass spectrometry analysis resulted in reliable identification of 50 proteins belonging to mitochondrial and extramitochondrial compartments. Since all these proteins were bound to avidin-agarose only after preincubation of the mitochondrial fraction with biotinylated ubiquitin, they could therefore be referred to as specifically bound proteins. A search for specific ubiquitination signature masses revealed several extramitochondrial and intramitochondrial ubiquitinated proteins representing about 20% of total number of proteins bound to avidin-agarose. The interactome analysis suggests that the identified non-ubiquitinated proteins obviously form tight complexes either with ubiquitinated proteins or with their partners and/or mitochondrial membrane components. Results of the present study demonstrate that the use of biotinylated ubiquitin may be considered as the method of choice for in vitro evaluation of endogenous ubiquitin-conjugating machinery in particular subcellular organelles and changes in ubiquitin/organelle associated interactomes. This may be useful for evaluation of changes in interactomes induced by protein ubiquitination under norm and various brain pathologies.
已研究了体外生物素化泛素在评估内源性泛素缀合及分析泛素相关蛋白质-蛋白质相互作用方面的适用性。用生物素化泛素孵育大鼠脑线粒体，随后在抗生物素蛋白-琼脂糖上进行亲和层析、充分洗涤、对结合在亲和吸附剂上的蛋白质进行胰蛋白酶消化并进行质谱分析，从而可靠地鉴定出了50种属于线粒体和线粒体外区室的蛋白质。由于所有这些蛋白质仅在将线粒体部分与生物素化泛素预孵育后才与抗生物素蛋白-琼脂糖结合，因此它们可被称为特异性结合蛋白。对特定泛素化特征质量的搜索揭示了几种线粒体外和线粒体内泛素化蛋白质，约占与抗生物素蛋白-琼脂糖结合的蛋白质总数的20%。相互作用组分析表明，所鉴定的非泛素化蛋白质显然与泛素化蛋白质或其伴侣和/或线粒体膜成分形成紧密复合物。本研究结果表明，生物素化泛素的使用可被视为体外评估特定亚细胞器中内源性泛素缀合机制以及泛素/细胞器相关相互作用组变化的首选方法。这对于评估正常和各种脑部病理情况下蛋白质泛素化诱导的相互作用组变化可能有用。
