Using Biotinylated SUMO-Traps to Analyze SUMOylated Proteins.

SUMO-interacting motifs (SIMs) recognize SUMOylated proteins with high specificity allowing to connect SUMO-modified proteins. Multiple SIMs fused to distinct tags have been used to increase their affinity and generate more efficient purification tools. Enrichment of SUMOylated proteins using SIMs arranged in tandem (SUMO-traps) facilitates the identification and characterization of protein targets in vitro and in vivo. Here a protocol to produce biotinylated SUMO-traps (bioSUBEs) to capture SUMO chains and typical SUMOylated proteins such as p53 or IkBα is presented. Biotinylated SUMO-traps represent an alternative to reduce the background associated to bigger tags, e.g., during mass spectrometry analysis. Consequently, bioSUBEs are alternative tools to characterize endogenous SUMO targets.

Proteostasis and SUMO in the heart.

Heart proteostasis relies on a complex and integrated network of molecular processes surveilling organ performance under physiological and pathological conditions. For this purpose, cardiac cells depend on the correct function of their proteolytic systems, such as the ubiquitin-proteasome system (UPS), autophagy and the calpain system. Recently, the role of protein SUMOylation (an ubiquitin-like modification), has emerged as important modulator of cardiac proteostasis, which will be the focus of this review.

Analysis of PTEN ubiquitylation and SUMOylation using molecular traps.

The function of the tumour suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is tightly controlled by post-translational modifications (PTMs) including ubiquitin or Small Ubiquitin-related MOdifiers (SUMO). It is known that SUMOylation by SUMO-1, SUMO-2/-3, mono- or polyubiquitylation have a distinct impact on PTEN activity, localisation and/or stability, however the molecular mechanisms governing these processes are still unclear. Studying PTM regulated events has always been a difficult task due to their labile nature. Here, we propose an update on the role of these PTMs on PTEN function, as well as a methodological overview on the use of molecular traps named SUMO Binding Entities (SUBEs) or Tandem Ubiquitin Binding Entities (TUBEs) to capture SUMOylated or Ubiquitylated forms of PTEN respectively. When combined with in vitro SUMOylation or Ubiquitylation assays, the use of molecular traps facilitate the detection of modified forms of PTEN. SUMO and ubiquitin-traps are also suitable to capture endogenously modified forms of PTEN after expression of E3 ligases or treatment with chemical inhibitors. This versatile approach represents an interesting alternative to explore PTEN regulation by SUMO and ubiquitin under physiological or pathological conditions.

Analysis of SUMOylated proteins using SUMO-traps.

SUMO-modified proteins are recognized by SUMO interacting motifs (SIMs), thus triggering diverse cellular responses. Here SIMs were used to develop SUMO-traps to capture endogenous SUMOylated proteins. Our results show that these small peptides are transferable motifs that maintain their SUMO binding capacity when fused to the heterologous carrier protein GST. The tandem disposition of SIMs increases the binding capacity of SUMO-traps to specifically interact with polySUMO but not poly-Ubiquitin chains. We demonstrate that this SUMO capturing system purifies SUMOylated proteins such as IκBα, PTEN, PML or p53 in vitro and in vivo. These properties can be used to explore the many critical functions regulated by protein SUMOylation.

Strategies to Identify Recognition Signals and Targets of SUMOylation.

SUMOylation contributes to the regulation of many essential cellular factors. Diverse techniques have been used to explore the functional consequences of protein SUMOylation. Most approaches consider the identification of sequences on substrates, adaptors, or receptors regulating the SUMO conjugation, recognition, or deconjugation. The large majority of the studied SUMOylated proteins contain the sequence [IVL]KxE. SUMOylated proteins are recognized by at least 3 types of hydrophobic SUMO-interacting motifs (SIMs) that contribute to coordinate SUMO-dependent functions. Typically, SIMs are constituted by a hydrophobic core flanked by one or two clusters of negatively charged amino acid residues. Multiple SIMs can integrate SUMO binding domains (SBDs), optimizing binding, and control over SUMO-dependent processes. Here, we present a survey of the methodologies used to study SUMO-regulated functions and provide guidelines for the identification of cis and trans sequences controlling SUMOylation. Furthermore, an integrative analysis of known and putative SUMO substrates illustrates an updated landscape of several SUMO-regulated events. The strategies and analysis presented here should contribute to the understanding of SUMO-controlled functions and provide rational approach to identify biomarkers or choose possible targets for intervention in processes where SUMOylation plays a critical role.

Role of monoubiquitylation on the control of IκBα degradation and NF-κB activity.

The NF-κB pathway is regulated by multiple post-translational modifications including phosphorylation, ubiquitylation and SUMOylation. Many of these modifications act on the natural inhibitor IκBα modulating its capacity to control signal-mediated NF-κB activity. While the canonical pathway involving the phosphorylation and polyubiquitylation of IκBα has been well characterized, the role of these post-translational modifications in the control of basal NF-κB activity has not been deeply explored. Using the recently developed Tandem-repeated Ubiquitin Binding Entities (also known as ubiquitin traps) to capture ubiquitylated proteins, we identified monoubiquitylated forms of IκBα from multiple rat organs and cell types. The identification of these forms was demonstrated through different procedures such as immunoprecipitations with specific ubiquitin antibodies or His6-Ubiquitin pull downs. Monoubiquitylated forms of IκBα are resistant to TNFα-mediated degradation and can be captured using TUBEs, even after proteasome inhibitors treatment. As it occurs for monoSUMOylation, monoubiquitylation is not dependent of the phosphorylation of IκBα on the serines 32/36 and is not optimally degraded after TNFα stimulation. A ubiquitin-IκBα fusion exhibits phosphorylation defects and resistance to TNFα mediated degradation similar to the ones observed for endogenous monoubiquitylated IκBα. The N-terminal attachment of a single ubiquitin moiety on the IκBα fusion results in a deficient binding to the IKKß kinase and recruitment of the SCF ligase component ßTrCP, promoting a negative impact on the NF-κB activity. Altogether, our results suggest the existence of a reservoir of monoubiquitylated IκBα resistant to TNFα-induced proteolysis, which is able to interact and repress DNA binding and NF-κB transcriptional activity. Such pool of IκBα may play an important role in the control of basal and signal-mediated NF-κB activity.