Purification and identification of endogenous polySUMO conjugates

Abstract

The small ubiquitin-like modifier (SUMO) can undergo selfmodification to form polymeric chains that have been implicated in cellular processes such as meiosis, genome maintenance and stress response. Investigations into the biological role of polymeric chains have been hampered by the absence of a protocol for the purification of proteins linked to SUMO chains. In this paper, we describe a rapid affinity purification procedure for the isolation of endogenous polySUMO-modified species that generates highly purified material suitable for individual protein studies and proteomic analysis. We use this approach to identify more than 300 putative polySUMO conjugates from cultured eukaryotic cells.

Regulation of USP28 Deubiquitinating Activity by SUMO Conjugation

“…Deubiquitinating Assays against Different Types of Ubiquitin Substrates-Human polyubiquitin chain (Lys 48 , Lys 63 , and 3-7ubs) and diubiquitin (Lys 48 and Lys 63 ) were purchased from Boston Biochem. They were dissolved in the buffer containing 250 mM NaCl, 20 nM Tris 8.0, and 1 mM ␤-mercaptoethanol with a final concentration of 1 mg/ml.…”

“…However, in contrast to USP25, the N-terminal region of USP28 does not impair the deubiquitinating activity of the catalytic domain, and the analysis of the SUMO2-USP28 fusion proteins suggests a direct interaction of SUMO with the catalytic domain of USP28. Interestingly, USP28 displays a chain preference for Lys 11 , Lys 48 , and Lys 63 diubiquitin linkages, in contrast to other nonspecific members of the USP deubiquitinase family. These results reveal unexpected diversity in the regulatory mechanisms and substrate preference of structurally similar USPs.…”

“…This post-translation modification consists of the formation of an isopeptidic bond between the C terminus of the Ub or Ubl molecule and a lysine residue of the target protein. Ubiquitin tagging is the major cellular signal to promote the proteasomal degradation of protein targets, normally by the formation of Lys 48 -linked polyubiquitin chains. In addition to the protein degradation pathway, Ubls have also been implicated in multiple cellular processes, mainly by regulating protein-protein interactions, protein localization, or enzymatic activity (3,4).…”

Poly-Small Ubiquitin-like Modifier (PolySUMO)-binding Proteins Identified through a String Search

“…Interestingly, RNF4 family proteins all contain multiple SIMs positioned closely as a cluster. This has raised the possibility that clustered SIMs are attuned to specifically recognize polySUMO chains (20,22). Although the significance of polysumoylation has been recognized, its function is yet to be well defined (23).…”

Large-scale analysis of lysine SUMOylation by SUMO remnant immunoaffinity profiling

“…Global or individual detection of endogenous SUMOylated substrates has been notoriously more challenging and was reported only in a limited number of cases. For example, Bruderer et al 25 have exploited the presence of 4 SUMOinteracting motif domains on the poly-SUMO-dependent ubiquitin E3 ligase RNF4 to identify more than 300 polySUMOylated proteins in HeLa cells following heat-shock treatment. A recent report described the use of commercial monoclonal antibodies to SUMO1 and SUMO2/3 for the analysis of individual SUMOylated proteins and global SUMO proteome analyses of mammalian cells and tissues 26 .…”