Ubiquitination and Degradation of Proteins
Modification by ubiquitin (Ub) and ubiquitin-like proteins (UbLs) is involved in the regulation of numerous cellular processes and has therefore become an important subject of research in various areas of biomedicine. The large number of components of the system (∼1,500), most of them being ligases (∼800) that recognize their target substrates specifically, along with the complexity of the ubiquitination process, mostly the synthesis of the hallmark polyubiquitin chains, has rendered studies of many of the processes related to the activity of the system resistant to detailed mechanistic analysis. Thus, our knowledge of the modes of recognition of target substrates by ligases and of consensus ubiquitination sites is sparse. We also lack basic tools such as antibodies directed against specific internal polyubiquitin chain linkages and analytical methods to decipher the structure of intact chains and their formation. All these tools are essential in order to understand the mechanisms that underlie the diverse activities of the system, proteolytic as well as non-proteolytic, and the manner in which it exerts its high specificity and selectivity toward its myriad substrates. Here we describe selected basic procedures that allow one to become acquainted with this rapidly evolving field, realizing that one cannot provide a comprehensive coverage of all or even a small part of the methodologies related to this research area. We provide information on how to set up a cell-free system for ubiquitination – a powerful tool that enables researchers to reconstitute the modification from purified components – and how to identify ubiquitin adducts in cells. Additionally, we describe methods to follow stability (degradation) of proteins in cell-free systems and in cells.
Key wordsUbiquitin ubiquitin aldehyde ubiquitination degradation reticulocyte lysate proteolytic substrates
Studies in the laboratory of AC are supported by the Angelman Syndrome Foundation (ASF) and by grants from the Dr. Miriam and Sheldon Adelson Foundation for Medical research (AMRF), the Israel Science Foundation (ISF), the German-Israeli Foundation for Research and Scientific Development (G.I.F.), the European Union (EU) Network of Excellence Rubicon, an Israel Cancer Research Fund (ICRF) USA Professorship, and a grant from the Foundation for Promotion of Research in the Technion.
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