Small Ubiquitin-like Modifier Chains Research Articles

Depolymerization of SUMO chains induces slender to stumpy differentiation in T. brucei bloodstream parasites.

Trypanosoma brucei are protozoan parasites that cause sleeping sickness in humans and nagana in cattle. Inside the mammalian host, a quorum sensing-like mechanism coordinates its differentiation from a slender replicative form into a quiescent stumpy form, limiting growth and activating metabolic pathways that are beneficial to the parasite in the insect host. The post-translational modification of proteins with the Small Ubiquitin-like MOdifier (SUMO) enables dynamic regulation of cellular metabolism. SUMO can be conjugated to its targets as a monomer but can also form oligomeric chains. Here, we have investigated the role of SUMO chains in T. brucei by abolishing the ability of SUMO to polymerize. We have found that parasites able to conjugate only SUMO monomers are primed for differentiation. This was demonstrated for monomorphic lines that are normally unable to produce stumpy forms in response to quorum sensing signaling in mice, and also for pleomorphic cell lines in which stumpy cells were observed at unusually low parasitemia levels. SUMO chain mutants showed a stumpy compatible transcriptional profile and better competence to differentiate into procyclics. Our study indicates that SUMO depolymerization may represent a coordinated signal triggered during stumpy activation program.

SUMO protease SENP6 protects the nucleus from hyperSUMOylation-induced laminopathy-like alterations

The small ubiquitin-like modifier (SUMO) protease SENP6 disassembles SUMO chains from cellular substrate proteins. We use a proteomic method to identify putative SENP6 substrates based on increased apparent molecular weight after SENP6 depletion. Proteins of the lamin family of intermediate filaments show substantially increased SUMO modification after SENP6 depletion. This is accompanied by nuclear structural changes remarkably like those associated with laminopathies. Two SUMO attachment sites on lamin A/C are close to sites of mutations in Emery-Driefuss and limb girdle muscular dystrophy. To establish a direct link between lamin SUMOylation and the observed phenotype, we developed proximity-induced SUMO modification (PISM), which fuses a lamin A/C targeting DARPin to a SUMO E3 ligase domain. This directly targets lamin A/C for SUMO conjugation and demonstrates that enhanced lamin SUMO modification recapitulates the altered nuclear structure manifest after SENP6 depletion. This shows SENP6 activity protects the nucleus against hyperSUMOylation-induced laminopathy-like alterations.

Insights into the Microscopic Structure of RNF4-SIM-SUMO Complexes from MD Simulations

Post-translational modification with one of the isoforms of the small ubiquitin-like modifier (SUMO) affects thousands of proteins in the human proteome. The binding of SUMO to SUMO interacting motifs (SIMs) can translate the SUMOylation event into functional consequences. The E3 ubiquitin ligase RNF4 contains multiple SIMs and connects SUMOylation to the ubiquitin pathway. SIM2 and SIM3 of RNF4 were shown to be the most important motifs to recognize SUMO chains. However, the study of SIM-SUMO complexes is complicated by their typically low affinity and variable binding of the SIMs in parallel and antiparallel orientations. We investigated properties of complexes formed by SUMO3 with peptides containing either SIM2 or SIM3 using molecular dynamics simulations. The affinities of the complexes were determined using a state-of-the-art free energy protocol and were found to be in good agreement with experimental data, thus corroborating our method. Long unrestrained simulations allowed a new interpretation of experimental results regarding the structure of the SIM-SUMO interface. We show that both SIM2 and SIM3 bind SUMO3 in parallel and antiparallel orientations and identified main interaction sites for acidic residues flanking the SIM. We noticed unusual SIM-SUMO interfaces in a previously reported NMR structure (PDB: 2mp2) of a complex formed by a SUMO3 dimer with the bivalent SIM2-SIM3 peptide. Computational determination of the individual SIM-SUMO affinities based on these structural arrangements yielded significantly higher dissociation constants. To our knowledge, our approach adds new opportunities to characterize individual SIM-SUMO complexes and suggests that further studies will be necessary to understand these interactions when occurring in multivalent form.

Revisiting the role of PML protein targeting and disruption of PML bodies in human cytomegalovirus infection

Promyelocytic leukaemia (PML) bodies are nuclear organelles implicated in post-translational modification by small ubiquitin-like modifier (SUMO) proteins and in the antiviral host cell response to infection. The 72-kDa immediate-early protein 1 (IE1) is considered the principal antagonist of PML bodies encoded by the human cytomegalovirus, one of eight human herpesviruses. Previous work has suggested that the interaction between IE1 and PML proteins, the central organisers of PML bodies, and the subsequent disruption of these organelles serve a critical role in viral replication by counteracting intrinsic antiviral immunity and the induction of interferon (IFN)-stimulated genes. However, this picture has emerged largely from studying mutant IE1 proteins known or predicted to be globally misfolded und metabolically unstable. We systematically screened for stable IE1 mutants by clustered charge-to-alanine scanning. We identified a mutant protein (IE1cc172-176) selectively defective for PML interaction. Functional comparisons between the mutant and wild-type protein revealed that IE1 can undergo modification by mixed polymeric SUMO chains and that it targets PML and Sp100, the two main constituents of PML bodies, via distinct mechanisms. Unexpectedly, IE1cc172-176 supported viral replication almost as efficiently as wild-type IE1. Moreover, lower instead of higher (as expected) levels of tumor necrosis factor alpha, IFN-beta, IFN-lambda and IFN-stimulated gene expression were observed with the mutant compared to the wild-type protein and virus. These results suggest that the disruption of PML bodies is linked to induction rather than inhibition of antiviral gene expression. Our findings challenge current views regarding the role of PML bodies in viral infection.

A FRET Sensor to Monitor Bivalent SUMO-SIM Interactions in SUMO Chain Binding.

The small ubiquitin-like modifier (SUMO) can be assembled into polymeric chains as part of its diverse biochemical signal pattern upon conjugation to substrate proteins. SUMO chain recognition is facilitated by receptor proteins that contain at least two SUMO-interacting motifs (SIMs). Little is known about the structure of SUMO chains, both in an unliganded form and upon complexation with multi-SIM protein partners. A FRET sensor has been developed based on a linear dimer of human SUMO-2 as a minimal SUMO chain analogue. The synthetic acceptor and donor dyes were conjugated by maleimide and copper-catalyzed click chemistry to each of the two SUMO subunits. FRET changes were only observed in the presence of di- or multi-SIM ligands. Alteration of the short linker sequence between SIMs 2 and 3 of RNF4 showed a great tolerance, and hence, structural flexibility, of the SUMO dimer for bivalent binding of adjacent SIMs. The di-SUMO FRET sensor reports on the binding of SIM clusters of the proteins C5orf25 and SOBP; this suggest that these can bind to adjacent subunits of a SUMO chain. The developed FRET sensor will be a useful tool to study the importance of SIM and linker sequences, as well as biochemical and structural properties of SUMO chains and multi-SIM proteins.

Site-specific inhibition of the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 selectively impairs SUMO chain formation

Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) regulate many cellular processes, including genome integrity, gene expression, and ribosome biogenesis. The E2-conjugating enzyme Ubc9 catalyzes the conjugation of SUMOs to ϵ-amino groups of lysine residues in target proteins. Attachment of SUMO moieties to internal lysines in Ubc9 itself can further lead to the formation of polymeric SUMO chains. Mono- and poly-SUMOylations of target proteins provide docking sites for distinct adapter and effector proteins important for regulating discrete SUMO-regulated pathways. However, molecular tools to dissect pathways depending on either mono- or poly-SUMOylation are largely missing. Using a protein-engineering approach, we generated high-affinity SUMO2 variants by phage display that bind the back side binding site of Ubc9 and function as SUMO-based Ubc9 inhibitors (SUBINs). Importantly, we found that distinct SUBINs primarily inhibit poly-SUMO chain formation, whereas mono-SUMOylation was not impaired. Proof-of-principle experiments demonstrated that in a cellular context, SUBINs largely prevent heat shock-triggered poly-SUMOylation. Moreover, SUBINs abrogated arsenic-induced degradation of promyelocytic leukemia protein. We propose that the availability of the new chain-selective SUMO inhibitors reported here will enable a thorough investigation of poly-SUMO-mediated cellular processes, such as DNA damage responses and cell cycle progression.

Evolution of SUMO Function and Chain Formation in Insects

SUMOylation, the covalent binding of Small Ubiquitin-like Modifier (SUMO) to target proteins, is a posttranslational modification that regulates critical cellular processes in eukaryotes. In insects, SUMOylation has been studied in holometabolous species, particularly in the dipteran Drosophila melanogaster, which contains a single SUMO gene (smt3). This has led to the assumption that insects contain a single SUMO gene. However, the analysis of insect genomes shows that basal insects contain two SUMO genes, orthologous to vertebrate SUMO1 and SUMO2/3. Our phylogenetical analysis reveals that the SUMO gene has been duplicated giving rise to SUMO1 and SUMO2/3 families early in Metazoan evolution, and that later in insect evolution the SUMO1 gene has been lost after the Hymenoptera divergence. To explore the consequences of this loss, we have examined the characteristics and different biological functions of the two SUMO genes (SUMO1 and SUMO3) in the hemimetabolous cockroach Blattella germanica and compared them with those of Drosophila Smt3. Here, we show that the metamorphic role of the SUMO genes is evolutionary conserved in insects, although there has been a regulatory switch from SUMO1 in basal insects to SUMO3 in more derived ones. We also show that, unlike vertebrates, insect SUMO3 proteins cannot form polySUMO chains due to the loss of critical lysine residues within the N-terminal part of the protein. Furthermore, the formation of polySUMO chains by expression of ectopic human SUMO3 has a deleterious effect in Drosophila. These findings contribute to the understanding of the functional consequences of the evolution of SUMO genes.

Structural insight into SUMO chain recognition and manipulation by the ubiquitin ligase RNF4.

The small ubiquitin-like modifier (SUMO) can form polymeric chains that are important signals in cellular processes such as meiosis, genome maintenance and stress response. The SUMO-targeted ubiquitin ligase RNF4 engages with SUMO chains on linked substrates and catalyses their ubiquitination, which targets substrates for proteasomal degradation. Here we use a segmental labelling approach combined with solution nuclear magnetic resonance (NMR) spectroscopy and biochemical characterization to reveal how RNF4 manipulates the conformation of the SUMO chain, thereby facilitating optimal delivery of the distal SUMO domain for ubiquitin transfer.

Visualizing and Quantifying Protein PolySUMOylation at the Single-Molecule Level

Protein polySUMOylation, the attachment of small ubiquitin-like modifier (SUMO) chains to the target protein, is associated with a variety of physiological processes. However, the analysis of protein polySUMOylation is often complicated by the heterogeneity of SUMO-target conjugates. Here, we develop a new strategy to visualize and quantify polySUMOylation at the single-molecule level by integrating the tetracysteine (TC) tag labeling technology and total internal reflection fluorescence (TIRF)-based single-molecule imaging. As a proof-of-concept, we employ the human SUMO-2 as the model. The addition of TC tag to SUMO-2 can specifically translate the SUMO-mediated modification into visible fluorescence signal without disturbing the function of SUMO-2. The SUMO monomers display homogeneous fluorescence spots at the single-molecule level, whereas the mixed SUMO chains exhibit nonuniform fluorescence spots with a wide range of intensities. Analysis of the number and the brightness of fluorescence spots enable quantitative measurement of the polySUMOylation degree inside the cells under different physiological conditions. Due to the frequent occurrence of posttranslational modification by polymeric chains in cells, this single-molecule strategy has the potential to be broadly applied for studying protein posttranslational modification in normal cellular physiology and disease etiology.

Targeted Identification of SUMOylation Sites in Human Proteins Using Affinity Enrichment and Paralog-specific Reporter Ions

Protein modification by small ubiquitin-like modifier (SUMO) modulates the activities of numerous proteins involved in different cellular functions such as gene transcription, cell cycle, and DNA repair. Comprehensive identification of SUMOylated sites is a prerequisite to determine how SUMOylation regulates protein function. However, mapping SUMOylated Lys residues by mass spectrometry (MS) is challenging because of the dynamic nature of this modification, the existence of three functionally distinct human SUMO paralogs, and the large SUMO chain remnant that remains attached to tryptic peptides. To overcome these problems, we created HEK293 cell lines that stably express functional SUMO paralogs with an N-terminal His6-tag and an Arg residue near the C terminus that leave a short five amino acid SUMO remnant upon tryptic digestion. We determined the fragmentation patterns of our short SUMO remnant peptides by collisional activation and electron transfer dissociation using synthetic peptide libraries. Activation using higher energy collisional dissociation on the LTQ-Orbitrap Elite identified SUMO paralog-specific fragment ions and neutral losses of the SUMO remnant with high mass accuracy (< 5 ppm). We exploited these features to detect SUMO modified tryptic peptides in complex cell extracts by correlating mass measurements of precursor and fragment ions using a data independent acquisition method. We also generated bioinformatics tools to retrieve MS/MS spectra containing characteristic fragment ions to the identification of SUMOylated peptide by conventional Mascot database searches. In HEK293 cell extracts, this MS approach uncovered low abundance SUMOylated peptides and 37 SUMO3-modified Lys residues in target proteins, most of which were previously unknown. Interestingly, we identified mixed SUMO-ubiquitin chains with ubiquitylated SUMO proteins (K20 and K32) and SUMOylated ubiquitin (K63), suggesting a complex crosstalk between these two modifications.

Abstract SY07-02: Involvement of ubiquitin ligase RNF4 in the DNA damage response

Abstract SY07-02: Involvement of ubiquitin ligase RNF4 in the DNA damage response

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.

Yeast axial-element protein, Red1, binds SUMO chains to promote meiotic interhomologue recombination and chromosome synapsis

The synaptonemal complex (SC) is a tripartite protein structure consisting of two parallel axial elements (AEs) and a central region. During meiosis, the SC connects paired homologous chromosomes, promoting interhomologue (IH) recombination. Here, we report that, like the CE component Zip1, Saccharomyces cerevisiae axial-element structural protein, Red1, can bind small ubiquitin-like modifier (SUMO) polymeric chains. The Red1–SUMO chain interaction is dispensable for the initiation of meiotic DNA recombination, but it is essential for Tel1- and Mec1-dependent Hop1 phosphorylation, which ensures IH recombination by preventing the inter-sister chromatid DNA repair pathway. Our results also indicate that Red1 and Zip1 may directly sandwich the SUMO chains to mediate SC assembly. We suggest that Red1 and SUMO chains function together to couple homologous recombination and Mec1–Tel1 kinase activation with chromosome synapsis during yeast meiosis.

Proteomics Analysis of Nucleolar SUMO-1 Target Proteins upon Proteasome Inhibition

Many cellular processes are regulated by the coordination of several post-translational modifications that allow a very fine modulation of substrates. Recently it has been reported that there is a relationship between sumoylation and ubiquitination. Here we propose that the nucleolus is the key organelle in which SUMO-1 conjugates accumulate in response to proteasome inhibition. We demonstrated that, upon proteasome inhibition, the SUMO-1 nuclear dot localization is redirected to nucleolar structures. To better understand this process we investigated, by quantitative proteomics, the effect of proteasome activity on endogenous nucleolar SUMO-1 targets. 193 potential SUMO-1 substrates were identified, and interestingly in several purified SUMO-1 conjugates ubiquitin chains were found to be present, confirming the coordination of these two modifications. 23 SUMO-1 targets were confirmed by an in vitro sumoylation reaction performed on nuclear substrates. They belong to protein families such as small nuclear ribonucleoproteins, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, histones, RNA-binding proteins, and transcription factor regulators. Among these, histone H1, histone H3, and p160 Myb-binding protein 1A were further characterized as novel SUMO-1 substrates. The analysis of the nature of the SUMO-1 targets identified in this study strongly indicates that sumoylation, acting in coordination with the ubiquitin-proteasome system, regulates the maintenance of nucleolar integrity.

Small Ubiquitin-like Modifier (SUMO) Modification of the Androgen Receptor Attenuates Polyglutamine-mediated Aggregation

The neurodegenerative disorder spinal and bulbar muscular atrophy or Kennedy disease is caused by a CAG trinucleotide repeat expansion within the androgen receptor (AR) gene. The resulting expanded polyglutamine tract in the N-terminal region of the receptor renders AR prone to ligand-dependent misfolding and formation of oligomers and aggregates that are linked to neuronal toxicity. How AR misfolding is influenced by post-translational modifications, however, is poorly understood. AR is a target of SUMOylation, and this modification inhibits AR activity in a promoter context-dependent manner. SUMOylation is up-regulated in response to multiple forms of cellular stress and may therefore play an important cytoprotective role. Consistent with this view, we find that gratuitous enhancement of overall SUMOylation significantly reduced the formation of polyglutamine-expanded AR aggregates without affecting the levels of the receptor. Remarkably, this effect requires SUMOylation of AR itself because it depends on intact AR SUMOylation sites. Functional analyses, however, indicate that the protective effects of enhanced AR SUMOylation are not due to alterations in AR transcriptional activity because a branched protein structure in the appropriate context of the N-terminal region of AR is necessary to antagonize aggregation but not for inhibiting AR transactivation. Remarkably, small ubiquitin-like modifier (SUMO) attenuates AR aggregation through a unique mechanism that does not depend on critical features essential for its interaction with canonical SUMO binding motifs. Our findings therefore reveal a novel function of SUMOylation and suggest that approaches that enhance AR SUMOylation may be of clinical use in polyglutamine expansion diseases.

Characterization of SUMO-conjugating enzyme mutants in Schizosaccharomyces pombe identifies a dominant-negative allele that severely reduces SUMO conjugation.

The phenotypes of mutants defective in the Schizosaccharomyces pombe SUMO (small, ubiquitin-like modifier)-conjugating enzyme Hus5 (the homologue of Ubc9) show that it is required for recovery from S-phase arrest. Unlike the case with ubiquitination, where ligases are required, SUMO-conjugating enzymes are sufficient for substrate recognition and conjugation of SUMO on to target proteins, at least in vitro. Thus SUMO-conjugating enzymes are likely to be important regulators of sumoylation. Here, we report on the characterization of two hus5 alleles. Although hus5.17 and hus5.62 respond in a similar manner to UV and ionizing radiation, they have different responses to the DNA-synthesis inhibitor, hydroxyurea. In addition, SUMO (Pmt3) is mislocalized in hus5.17 cells, but not in hus5.62 mutant cells. The mutations in hus5.62 and hus5.17 map to Ala(129) and the 5' splice site of intron 2 respectively. We have characterized the Hus5.62 protein and shown, in vitro, that it still interacts with SUMO and at least one protein, Rad22, which is a SUMO-modified target. The Hus5.62 protein is also capable of forming a thioester link with SUMO, but it does not function in sumoylation assays, either in the modification of Rad22 or in SUMO chain formation. When overexpressed in wild-type S. pombe cells, the Hus5.62 protein has a dominant-negative effect on sumoylation.