Michael H. Tatham

Polymeric Chains of SUMO-2 and SUMO-3 Are Conjugated to Protein Substrates by SAE1/SAE2 and Ubc9

Conjugation of the small ubiquitin-like modifier SUMO-1/SMT3C/Sentrin-1 to proteins in vitro is dependent on a heterodimeric E1 (SAE1/SAE2) and an E2 (Ubc9). Although SUMO-2/SMT3A/Sentrin-3 and SUMO-3/SMT3B/Sentrin-2 share 50% sequence identity with SUMO-1, they are functionally distinct. Inspection of the SUMO-2 and SUMO-3 sequences indicates that they both contain the sequence ψKXE, which represents the consensus SUMO modification site. As a consequence SAE1/SAE2 and Ubc9 catalyze the formation of polymeric chains of SUMO-2 and SUMO-3 on protein substrates in vitro, and SUMO-2 chains are detectedin vivo. The ability to form polymeric chains is not shared by SUMO-1, and although all SUMO species use the same conjugation machinery, modification by SUMO-1 and SUMO-2/-3 may have distinct functional consequences.

RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation

In acute promyelocytic leukaemia (APL), the promyelocytic leukaemia (PML) protein is fused to the retinoic acid receptor α (RAR). This disease can be treated effectively with arsenic, which induces PML modification by small ubiquitin-like modifiers (SUMO) and proteasomal degradation. Here we demonstrate that the RING-domain-containing ubiquitin E3 ligase, RNF4 (also known as SNURF), targets poly-SUMO-modified proteins for degradation mediated by ubiquitin. RNF4 depletion or proteasome inhibition led to accumulation of mixed, polyubiquitinated, poly-SUMO chains. PML protein accumulated in RNF4-depleted cells and was ubiquitinated by RNF4 in a SUMO-dependent fashion in vitro. In the absence of RNF4, arsenic failed to induce degradation of PML and SUMO-modified PML accumulated in the nucleus. These results demonstrate that poly-SUMO chains can act as discrete signals from mono-SUMOylation, in this case targeting a poly-SUMOylated substrate for ubiquitin-mediated proteolysis.

System-Wide Changes to SUMO Modifications in Response to Heat Shock

The small ubiquitin-like modifier protein SUMO is redistributed among many targets to mediate both short- and long-term signaling events. SUMO Status Revealed Posttranslational modification of proteins through their conjugation to small ubiquitin-like modifier (SUMO) proteins is important in the nucleus for the repair of damaged DNA and the maintenance of chromosome structure, as well as for a number of cytoplasmic processes. Although the machinery involved in attaching SUMO moieties to target proteins is well characterized, less is known about the upstream signals that trigger this modification. Golebiowski et al. designed a highly stringent, quantitative approach, involving protein purification and mass spectrometric techniques, to perform a system-wide analysis of the SUMOylation states of hundreds of proteins in HeLa cells in response to heat shock. The authors also analyzed the dynamic nature of SUMOylation in cells during the subsequent recovery phase. In addition to identifying many previously unknown substrates of SUMO-2, this proteome-wide analysis of SUMOylation revealed a rapid and dramatic redistribution of SUMO-2 among proteins involved in short- or long-term responses to heat stress. This new approach should also prove valuable in systems-wide analysis of other posttranslational modifications. Covalent conjugation of the small ubiquitin-like modifier (SUMO) proteins to target proteins regulates many important eukaryotic cellular mechanisms. Although the molecular consequences of the conjugation of SUMO proteins are relatively well understood, little is known about the cellular signals that regulate the modification of their substrates. Here, we show that SUMO-2 and SUMO-3 are required for cells to survive heat shock. Through quantitative labeling techniques, stringent purification of SUMOylated proteins, advanced mass spectrometric technology, and novel techniques of data analysis, we quantified heat shock–induced changes in the SUMOylation state of 766 putative substrates. In response to heat shock, SUMO was polymerized into polySUMO chains and redistributed among a wide range of proteins involved in cell cycle regulation; apoptosis; the trafficking, folding, and degradation of proteins; transcription; translation; and DNA replication, recombination, and repair. This comprehensive proteomic analysis of the substrates of a ubiquitin-like modifier (Ubl) identifies a pervasive role for SUMO proteins in the biologic response to hyperthermic stress.

Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis

Ubiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING domain of rat RNF4 in complex with E2 (UbcH5A) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The carboxy-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilize the consequent tetrahedral transition-state intermediate.

In Vivo Identification of Human Small Ubiquitin-like Modifier Polymerization Sites by High Accuracy Mass Spectrometry and an in Vitro to in Vivo Strategy

The length and precise linkage of polyubiquitin chains is important for their biological activity. Although other ubiquitin-like proteins have the potential to form polymeric chains their identification in vivo is challenging and their functional role is unclear. Vertebrates express three small ubiquitin-like modifiers, SUMO-1, SUMO-2, and SUMO-3. Mature SUMO-2 and SUMO-3 are nearly identical and contain an internal consensus site for sumoylation that is missing in SUMO-1. Combining state-of-the-art mass spectrometry with an “in vitro to in vivo” strategy for post-translational modifications, we provide direct evidence that SUMO-1, SUMO-2, and SUMO-3 form mixed chains in cells via the internal consensus sites for sumoylation in SUMO-2 and SUMO-3. In vitro, the chain length of SUMO polymers could be influenced by changing the relative amounts of SUMO-1 and SUMO-2. The developed methodology is generic and can be adapted for the identification of other sumoylation sites in complex samples.

Purification and identification of endogenous polySUMO conjugates

The small ubiquitin‐like modifier (SUMO) can undergo self‐modification 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.

Comparative Proteomic Analysis Identifies a Role for SUMO in Protein Quality Control

Changes in cellular SUMOylation occur in response to the accumulation of misfolded proteins. SUMO and the Proteasome Members of the family of ubiquitin-like posttranslational protein modifiers, such as small ubiquitin-like modifiers (SUMOs), share structural similarity with ubiquitin and form conjugates with target proteins to regulate many eukaryotic cellular functions. Noting that inhibition of the proteasome results in the accumulation of SUMO-conjugated proteins, Tatham et al. used mass spectrometric analysis of purified proteins to perform a system-wide analysis of protein SUMOylation in HeLa cells exposed to the proteasomal inhibitor MG132. Changes in SUMOylation were similar to those known to be triggered by heat stress and seemed to be secondary to the accumulation of misfolded proteins. In addition, SUMO-2 and ubiquitin were exchanged among substrates during proteasome inhibition. Together, these findings suggest SUMO proteins are involved in the response to the accumulation of misfolded proteins in cells. The small ubiquitin-like modifiers (SUMOs) alter the functions of diverse cellular proteins by covalent posttranslational modification and thus influence many cellular functions, including gene transcription, cell cycle, and DNA repair. Although conjugation by ubiquitin and SUMO-2/3 are largely functionally and mechanistically independent from one another, both appear to increase under conditions of proteasome inhibition. To better understand the relationship between SUMO and protein degradation by the proteasome, we performed a quantitative proteomic analysis of SUMO-2 substrates after short- and long-term inhibition of the proteasome with MG132. Comparisons with changes to the SUMO-2 conjugate subproteome in response to heat stress revealed qualitative and quantitative parallels between both conditions; however, in contrast to heat stress, the MG132-triggered increase in SUMO-2 conjugation depended strictly on protein synthesis, implying that the accumulation of newly synthesized, misfolded proteins destined for degradation by the proteasome triggered the SUMO conjugation response. Furthermore, proteasomal inhibition resulted in the accumulation of conjugated forms of all SUMO paralogs in insoluble protein inclusions and in the accumulation on SUMO-2 substrates of lysine-63–linked polyubiquitin chains, which are not thought to serve as signals for proteasome-mediated degradation. Together, these findings suggest multiple, proteasome-independent roles for SUMOs in the cellular response to the accumulation of misfolded proteins.

Unique binding interactions among Ubc9, SUMO and RanBP2 reveal a mechanism for SUMO paralog selection

The conjugation of small ubiquitin-like modifiers SUMO-1, SUMO-2 and SUMO-3 onto target proteins requires the concerted action of the specific E1-activating enzyme SAE1/SAE2, the E2-conjugating enzyme Ubc9, and an E3-like SUMO ligase. NMR chemical shift perturbation was used to identify the surface of Ubc9 that interacts with the SUMO ligase RanBP2. Unlike known ubiquitin E2-E3 interactions, RanBP2 binds to the β-sheet of Ubc9. Mutational disruption of Ubc9-RanBP2 binding affected SUMO-2 but not SUMO-1 conjugation to Sp100 and to a newly identified RanBP2 substrate, PML. RanBP2 contains a binding site specific for SUMO-1 but not SUMO-2, indicating that a Ubc9–SUMO-1 thioester could be recruited to RanBP2 via SUMO-1 in the absence of strong binding between Ubc9 and RanBP2. Thus we show that E2-E3 interactions are not conserved across the ubiquitin-like protein superfamily and identify a RanBP2-dependent mechanism for SUMO paralog–specific conjugation.

Proteome-Wide Identification of SUMO2 Modification Sites

A novel method for the enrichment of SUMO2-modified peptides reveals more than 1000 sites of modification in human cells. Sleuthing SUMO Sites Covalent attachment to small ubiquitin-like modifiers (SUMOs) alters the stability, localization, and function of diverse proteins. SUMO-specific enzymes transfer SUMO to lysines on target proteins in a process known as sumoylation. Tammsalu et al. present a method to discover the exact sites to which SUMOs conjugate. Overexpression of a mutated form of SUMO2 and subsequent cleavage with a sequence-specific endoproteinase resulted in tagged sumoylated peptides. Antibody-based enrichment of tagged peptides and analyses by proteomics identified more than 1000 sumoylated sites in the human proteome, thus providing a wealth of information for future studies. Posttranslational modification with small ubiquitin-like modifiers (SUMOs) alters the function of proteins involved in diverse cellular processes. SUMO-specific enzymes conjugate SUMOs to lysine residues in target proteins. Although proteomic studies have identified hundreds of sumoylated substrates, methods to identify the modified lysines on a proteomic scale are lacking. We developed a method that enabled proteome-wide identification of sumoylated lysines that involves the expression of polyhistidine (6His)–tagged SUMO2 with Thr90 mutated to Lys. Endoproteinase cleavage with Lys-C of 6His-SUMO2T90K–modified proteins from human cell lysates produced a diGly remnant on SUMO2T90K-conjugated lysines, enabling immunoprecipitation of SUMO2T90K–modified peptides and producing a unique mass-to-charge signature. Mass spectrometry analysis of SUMO-enriched peptides revealed more than 1000 sumoylated lysines in 539 proteins, including many functionally related proteins involved in cell cycle, transcription, and DNA repair. Not only can this strategy be used to study the dynamics of sumoylation and other potentially similar posttranslational modifications, but also, these data provide an unprecedented resource for future research on the role of sumoylation in cellular physiology and disease.

Detection of protein SUMOylation in vivo

The small ubiquitin-like modifiers (SUMOs) are posttranslationally conjugated to eukaryotic cellular proteins with generally unpredictable consequences. SUMO substrates are found in many cellular systems, and functional analysis has revealed that substrate SUMOylation often has an important role in their regulation. Here we describe a cell-based protocol which can be used to detect the SUMOylation of a protein that relies on the enrichment of SUMO conjugates by purification of 6His-SUMO under denaturing conditions, followed by western blotting for the protein of interest. By purifying under denaturing conditions this method not only reduces the risk of false-positive identifications by non-covalent interactions, but also preserves SUMO-substrate conjugates by inhibiting SUMO-specific proteases—two caveats that may complicate other less stringent purification methods. In preliminary form, this protocol takes 4–5 d to perform, and it can be further elaborated to provide a multi-angled approach to investigate protein conjugation by SUMO.