Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities

AUTOR(ES)

Hjerpe, Roland

FONTE

Nature Publishing Group

RESUMO

Post-translational modification with ubiquitin is one of the most important mechanisms in the regulation of protein stability and function. However, the high reversibility of this modification is the main obstacle for the isolation and characterization of ubiquitylated proteins. To overcome this problem, we have developed tandem-repeated ubiquitin-binding entities (TUBEs) based on ubiquitin-associated (UBA) domains. TUBEs recognize tetra-ubiquitin with a markedly higher affinity than single UBA domains, allowing poly-ubiquitylated proteins to be efficiently purified from cell extracts in native conditions. More significant is the fact that TUBEs protect poly-ubiquitin-conjugated proteins, such as p53 and IκBα, both from proteasomal degradation and de-ubiquitylating activity present in cell extracts, as well as from existing proteasome and cysteine protease inhibitors. Therefore, these new ‘molecular traps' should become valuable tools for purifying endogenous poly-ubiquitylated proteins, thus contributing to a better characterization of many essential functions regulated by these post-translational modifications.

Documentos Relacionados

• Regulation of the RSP5 Ubiquitin Ligase by an Intrinsic Ubiquitin-binding Site*
• A ubiquitin-binding motif required for intramolecular monoubiquitylation, the CUE domain
• NEMO specifically recognizes K63-linked poly-ubiquitin chains through a new bipartite ubiquitin-binding domain
• HSP27 Is a Ubiquitin-Binding Protein Involved in I-κBα Proteasomal Degradation
• Purification of DNA-binding proteins using tandem DNA-affinity column.