Functional profiling of endogenous SUMOylation sites

Abstract

Small ubiquitin-like modifier (SUMO) post-translational modification regulates numerous cellular processes. Ubiquitylated proteins have previously been profiled by treating with trypsin to generate a diglycl-lysine (KGG) motif from the carboxy-terminus of ubiquitin, which can be detected through mass spectrometry. Unlike ubiquitin, detection of endogenous SUMOylated proteins is limited by the lack of naturally occurring protease sites in the tail of SUMO proteins. We recently reported a method for proteome-wide, site-level detection of endogenous SUMOylation that uses α-lytic protease, WaLP, which generates peptides containing SUMO-remnant KGG at the site of SUMO modification. Using previously developed immuno-affinity isolation of KGG-containing peptides followed by mass spectrometry, this method allows for the parallel identification and quantitation of endogenous ubiquitylated and SUMOylated peptides. We identified 1209 unique endogenous SUMO modification sites, of which 826 had not been previously reported. While MG132 inhibition of the proteasome altered the abundance of both ubiquitylated and sumoylated proteins, only 413 of the SUMO sites identified matched previously known sites of ubiquitylation. Gene ontology of the ubiquitylated proteins has revealed diverse localization and functionality; ubiquitylated proteins are broadly involved in protein or DNA binding, cell cycle regulation, translocation, and the proteasome while SUMOylated proteins localize to the nucleus and are directly involved in nucleotide binding or processing. Eight of the ten most abundant SUMOylated proteins in the dataset are directly involved in nuclear translocation, DNA binding or transcription, and of the 83 unique SUMOylation sites for those proteins, we identified 27 previously unreported sites. The most abundant protein modified by SUMO or ubiquitin was ubiquitin itself, and the presence of multiply-modified peptides suggests that both can adopt branched-chain architectures. Our findings demonstrate that SUMO has a specialized function in nucleotide processing alongside the more diverse ubiquitylation pathway while maintaining a limited cross-talk between the two. Support or Funding Information This work was supported by NSF MCB 1244506 (E.A.K.) and NIH T32 GM 008326 (R.J.L.). Illustration of a recently reported method for parallel identification of SUMOylation and ubiquitylation This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.