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Control APC interactome network

Proteins measured by APC affinity enrichment-mass spectrometry and enrichment analysis of APC interactors

Acute Promyelocytic Leukemia is caused by expression of the oncogenic Promyelocytic Leukemia (PML)–Retinoic Acid Receptor Alpha (RARA) fusion protein. Therapy with arsenic trioxide results in degradation of PML-RARA and PML and cures the disease. Modification of PML and PML-RARA with SUMO and ubiquitin precedes ubiquitin-mediated proteolysis. To identify additional components of this pathway, we performed proteomics on PML bodies. This revealed that association of p97/VCP segregase with PML bodies is increased after arsenic treatment. Pharmacological inhibition of p97 altered the number, morphology, and size of PML bodies, accumulated SUMO and ubiquitin modified PML and blocked arsenic-induced degradation of PML-RARA and PML. p97 localized to PML bodies in response to arsenic, and siRNA-mediated depletion showed that p97 cofactors UFD1 and NPLOC4 were critical for PML degradation. Thus, the UFD1-NPLOC4-p97 segregase complex is required to extract poly-ubiquitinated, poly-SUMOylated PML from PML bodies, prior to degradation by the proteasome.

Arsenic is an effective treatment for Acute Promyelocytic Leukaemia as it induces degradation of the Promyelocytic Leukaemia (PML) – retinoic acid receptor alpha (RARA) oncogenic fusion protein. Some patients relapse with arsenic resistant disease because of missense mutations in PML. To determine the mechanistic basis for arsenic resistance we reconstituted PML-/- cells with YFP fusions of wild type (WT) and two mutant forms of PMLV found in patients refractory to arsenic, A216T and L217F. Both mutants formed PML bodies that were larger, but less numerous than WT and neither responded to arsenic by degradation. Analysis of immunoprecipitated PML bodies indicated that while WT PML experiences increased SUMO1, SUMO2/3 and ubiquitin conjugation, A216T PML is almost completely unresponsive and therefore does not recruit the SUMO targeted ubiquitin E3 ligase RNF4. Compared to WT PML, L217F PML was modified to a similar extent by SUMO2 but not SUMO1 and although it recruited RNF4, it failed to develop the appropriate poly-ubiquitin conjugates required to recruit the segregase p97, which is essential for PML degradation.

In terms of its relative frequency, lysine is a common amino acid in the human proteome. However, by bioinformatics we find hundreds of proteins that contain long and evolutionarily conserved stretches completely devoid of lysine residues. These so-called lysine deserts show a high prevalence in intrinsically disordered proteins with known or predicted functions within the ubiquitin-proteasome system (UPS), including many E3 ubiquitin-protein ligases and UBL domain proteasome substrate shuttles, such as BAG6, RAD23A, UBQLN1 and UBQLN2. We show that introduction of lysine residues into the deserts leads to a striking increase in ubiquitylation of some of these proteins. In case of BAG6, we show that ubiquitylation is catalyzed by the E3 RNF126, while RAD23A is ubiquitylated by E6AP. Despite the elevated ubiquitylation, mutant RAD23A appears stable, but displays a partial loss of function phenotype in fission yeast. In case of UBQLN1 and BAG6, introducing lysine leads to a reduced abundance due to proteasomal degradation of the proteins. For UBQLN1 we show that arginine residues within the lysine depleted region are critical for its ability to form cytosolic speckles/inclusions. We propose that selective pressure to avoid lysine residues may be a common evolutionary mechanism to prevent unwarranted ubiquitylation and/or perhaps other lysine post-translational modifications. This may be particularly relevant for UPS components as they closely and frequently encounter the ubiquitylation machinery and are thus more susceptible to nonspecific ubiquitylation.

Significance Primary host defenses against viruses involve specific cellular recognition of non-self nucleic acids as pathogen-associated molecular patterns (PAMPs) that trigger induction of cytokine-mediated antiviral responses. Thus, ability to discriminate between “self” and “non-self” nucleic acids, and prevent aberrant immunopathology, is a key tenet of immunity. Here, we identify self-derived endogenous retroviral RNAs as host-encoded PAMPs that are up-regulated during influenza virus infections, and which stimulate antiviral immunity. Normally, endogenous retroviruses are tightly repressed transcriptionally by host TRIM28, but infection triggers changes in the modification status of TRIM28 to alleviate repression. This provides an example of how endogenous retroviruses integrated within the host genome have been functionally co-opted by a regulatory switch to aid defense against newly invading pathogens., Dynamic small ubiquitin-like modifier (SUMO) linkages to diverse cellular protein groups are critical to orchestrate resolution of stresses such as genome damage, hypoxia, or proteotoxicity. Defense against pathogen insult (often reliant upon host recognition of “non-self” nucleic acids) is also modulated by SUMO, but the underlying mechanisms are incompletely understood. Here, we used quantitative SILAC-based proteomics to survey pan-viral host SUMOylation responses, creating a resource of almost 600 common and unique SUMO remodeling events that are mounted during influenza A and B virus infections, as well as during viral innate immune stimulation. Subsequent mechanistic profiling focused on a common infection-induced loss of the SUMO-modified form of TRIM28/KAP1, a host transcriptional repressor. By integrating knockout and reconstitution models with system-wide transcriptomics, we provide evidence that influenza virus-triggered loss of SUMO-modified TRIM28 leads to derepression of endogenous retroviral (ERV) elements, unmasking this cellular source of “self” double-stranded (ds)RNA. Consequently, loss of SUMO-modified TRIM28 potentiates canonical cytosolic dsRNA-activated IFN-mediated defenses that rely on RIG-I, MAVS, TBK1, and JAK1. Intriguingly, although wild-type influenza A virus robustly triggers this SUMO switch in TRIM28, the induction of IFN-stimulated genes is limited unless expression of the viral dsRNA-binding protein NS1 is abrogated. This may imply a viral strategy to antagonize such a host response by sequestration of induced immunostimulatory ERV dsRNAs. Overall, our data reveal that a key nuclear mechanism that normally prevents aberrant expression of ERV elements (ERVs) has been functionally co-opted via a stress-induced SUMO switch to augment antiviral immunity.

Macroautophagy and the ubiquitin proteasome system work as an interconnected network in the maintenance of cellular homeostasis. Indeed, efficient activation of macroautophagy upon nutritional deprivation is sustained by degradation of preexisting proteins by the proteasome. However, the specific substrates that are degraded by the proteasome in order to activate macroautophagy are currently unknown. By quantitative proteomic analysis we identified several proteins downregulated in response to starvation but independently of ATG5 expression. Among them, the most significant was HERPUD1, an ER protein of short-half life and a well-known substrate of the proteasome. We found that increased HERPUD1 stability by deletion of its ubiquitin-like domain (UBL) plays a negative role on basal and induced macroautophagy. Moreover, we found it triggers ER expansion by reordering the ER in crystalloid structures, but in the absence of unfolded protein response activation. Surprisingly, we found ER expansion led to an increase in the number and function of lysosomes establishing a tight network with the presence of membrane-contact sites. Importantly, a phosphomimetic S59D mutation within the UBL mimics UBL deletion on its stability and the ER-lysosomal network expansion revealing an increase of cell survival under stress conditions. Altogether, we propose stabilized HERPUD1 downregulates macroautophagy favoring instead a closed interplay between the ER and lysosomes with consequences in cell stress survival. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/447273v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@19ac57eorg.highwire.dtl.DTLVardef@1c095corg.highwire.dtl.DTLVardef@aa8fb1org.highwire.dtl.DTLVardef@1bfadce_HPS_FORMAT_FIGEXP M_FIG C_FIG

Su et al. show that SUMOylation stabilizes bioriented kinetochore–microtubule attachments in mitosis to allow timely anaphase onset. They identify the shugoshin pericentromeric adaptor protein and the chromosome passenger complex as SUMOylation targets, which lead to dampening of error correction pathways., During mitosis, sister chromatids attach to microtubules from opposite poles, called biorientation. Sister chromatid cohesion resists microtubule forces, generating tension, which provides the signal that biorientation has occurred. How tension silences the surveillance pathways that prevent cell cycle progression and correct erroneous kinetochore–microtubule attachments remains unclear. Here we show that SUMOylation dampens error correction to allow stable sister kinetochore biorientation and timely anaphase onset. The Siz1/Siz2 SUMO ligases modify the pericentromere-localized shugoshin (Sgo1) protein before its tension-dependent release from chromatin. Sgo1 SUMOylation reduces its binding to protein phosphatase 2A (PP2A), and weakening of this interaction is important for stable biorientation. Unstable biorientation in SUMO-deficient cells is associated with persistence of the chromosome passenger complex (CPC) at centromeres, and SUMOylation of CPC subunit Bir1 also contributes to timely anaphase onset. We propose that SUMOylation acts in a combinatorial manner to facilitate dismantling of the error correction machinery within pericentromeres and thereby sharpen the metaphase–anaphase transition.

To determine the role of SUMO modification in the maintenance of pluripotent stem cells we used ML792, a potent and selective inhibitor of SUMO Activating Enzyme. Treatment of human induced pluripotent stem cells with ML792 initiated changes associated with loss of pluripotency such as reduced expression of key pluripotency markers. To identify putative effector proteins and establish sites of SUMO modification, cells were engineered to stably express either SUMO1 or SUMO2 with TGG to KGG mutations that facilitate GlyGly-K peptide immunoprecipitation and identification. A total of 976 SUMO sites were identified in 427 proteins. STRING enrichment created 3 networks of proteins with functions in regulation of gene expression, ribosome biogenesis and RNA splicing, although the latter two categories represented only 5% of the total GGK peptide intensity. The remainder have roles in transcription and chromatin structure regulation. Many of the most heavily SUMOylated proteins form a network of zinc-finger transcription factors centred on TRIM28 and associated with silencing of retroviral elements. At the level of whole proteins there was only limited evidence for SUMO paralogue-specific modification, although at the site level there appears to be a preference for SUMO2 modification over SUMO1 in acidic domains. We show that SUMO is involved in the maintenance of the pluripotent state in hiPSCs and identify many chromatin-associated proteins as bona fide SUMO substrates in human induced pluripotent stem cells.

Pluripotent stem cells represent a powerful system to identify the mechanisms governing cell fate decisions during early mammalian development. Covalent attachment of the Small Ubiquitin Like Modifier (SUMO) to proteins has emerged as an important factor in stem cell maintenance. Here we show that SUMO is required to maintain stem cells in their pluripotent state and identify many chromatin-associated proteins as bona fide SUMO substrates in human induced pluripotent stem cells (hiPSCs). Loss of SUMO increases chromatin accessibility and expression of long non-coding RNAs and human endogenous retroviral elements, indicating a role for the SUMO modification of SETDB1 and a large TRIM28 centric network of zinc finger proteins in silencing of these elements. While most protein coding genes are unaffected, the Preferentially Expressed Antigen of Melanoma (PRAME) gene locus becomes more accessible and transcription is dramatically increased after inhibition of SUMO modification. When PRAME is silent, a peak of SUMO over the transcriptional start site overlaps with ChIP-seq peaks for cohesin, RNA pol II, CTCF and ZNF143, with the latter two heavily modified by SUMO. These associations suggest that silencing of the PRAME gene is maintained by the influence of SUMO on higher order chromatin structure. Our data indicate that SUMO modification plays an important role in hiPSCs by repressing genes that disrupt pluripotency networks or drive differentiation.

Summary To understand gene function, the encoding DNA or mRNA transcript can be manipulated and the consequences observed. However, these approaches do not have a direct effect on the protein product of the gene, which is either permanently abrogated or depleted at a rate defined by the half-life of the protein. We therefore developed a single-component system that could induce the rapid degradation of the specific endogenous protein itself. A construct combining the RING domain of ubiquitin E3 ligase RNF4 with a protein-specific camelid nanobody mediates target destruction by the ubiquitin proteasome system, a process we describe as antibody RING-mediated destruction (ARMeD). The technique is highly specific because we observed no off-target protein destruction. Furthermore, bacterially produced nanobody-RING fusion proteins electroporated into cells induce degradation of target within minutes. With increasing availability of protein-specific nanobodies, this method will allow rapid and specific degradation of a wide range of endogenous proteins., Graphical Abstract, Highlights • Antibody RING-mediated destruction (ARMeD) targets endogenous proteins for degradation • ARMeD is mediated by a nanobody fused to the RING domain of ubiquitin E3 ligase RNF4 • Nanobody-RING fusions introduced into cells degrade target proteins within minutes, To study gene function, a single-component system that induces rapid degradation of the protein product of the gene was developed. Described as antibody RING-mediated destruction (ARMeD), a nanobody fused to the RING domain of ubiquitin E3 ligase RNF4 mediates degradation of the target protein by the ubiquitin proteasome system.

To investigate the role of SUMO modification in the maintenance of pluripotent stem cells, we used ML792, a potent and selective inhibitor of SUMO Activating Enzyme. Treatment of human induced pluripotent stem cells with ML792 resulted in the loss of key pluripotency markers. To identify putative effector proteins and establish sites of SUMO modification, cells were engineered to stably express either SUMO1 or SUMO2 with C-terminal TGG to KGG mutations that facilitate GlyGly-K peptide immunoprecipitation and identification. A total of 976 SUMO sites were identified in 427 proteins. STRING enrichment created three networks of proteins with functions in regulation of gene expression, ribosome biogenesis, and RNA splicing, although the latter two categories represented only 5% of the total GGK peptide intensity. The rest have roles in transcription and the regulation of chromatin structure. Many of the most heavily SUMOylated proteins form a network of zinc-finger transcription factors centered on TRIM28 and associated with silencing of retroviral elements. At the level of whole proteins, there was only limited evidence for SUMO paralogue-specific modification, although at the site level there appears to be a preference for SUMO2 modification over SUMO1 in acidic domains. We show that SUMO influences the pluripotent state in hiPSCs and identify many chromatin-associated proteins as bona fide SUMO substrates in human induced pluripotent stem cells., Graphical Abstract, Highlights • SUMO inhibition in human stem cells reduced pluripotency markers. • SUMO site proteomics identified 976 SUMO sites in 427 proteins. • SUMO targets functioned in gene expression, ribosome biogenesis, and RNA splicing. • There is a preference for SUMO2 over SUMO1 modification in acidic domains., In Brief The role of SUMO modification in maintenance of human induced pluripotent stem cells was investigated using an inhibitor of SUMO modification. Key markers of pluripotency were lost after inhibitor treatment. Employing SUMO site proteomics, we identified 976 sites in 427 proteins. A major network of zinc-finger transcription factors linked to TRIM28 was associated with silencing of retroviral elements. At the site level there appears to be a preference for SUMO2 modification over SUMO1 in acidic domains.

SummaryAdenomatous polyposis coli(APC) is the most frequently mutated gene in colorectal cancer. APC negatively regulates the pro-proliferative Wnt signaling pathway by promoting the degradation of β-catenin, but the extent to which APC exerts Wnt/β-catenin-independent tumor suppressive activity is unclear. To identify interaction partners and β-catenin-independent targets of endogenous, full-length APC, we applied label-free and multiplexed TMT mass spectrometry. Affinity enrichment-mass spectrometry revealed over 150 previously unidentified APC interaction partners. Moreover, our global proteomic analysis revealed that roughly half of the protein expression changes that occur in response to APC loss are independent of β-catenin. By combining these two analyses, we identified Misshapen-like kinase 1 (MINK1) as a putative substrate of an alternative APC-containing destruction complex and provide evidence for the potential contribution of MINK1 toAPCmutant phenotypes. Collectively, our results highlight the extent and importance of Wnt-independent APC functions in epithelial biology and disease.

Adenomatous polyposis coli (APC) is the most frequently mutated gene in colorectal cancer. APC negatively regulates the pro-proliferative Wnt signaling pathway by promoting the degradation of &#946-catenin, but the extent to which APC exerts Wnt/&#946-catenin-independent tumor suppressive activity is unclear. To identify interaction partners and &#946-catenin-independent targets of endogenous, full-length APC, we applied label-free and multiplexed TMT mass spectrometry. Affinity enrichment-mass spectrometry revealed over 150 previously unidentified APC interaction partners. Moreover, our global proteomic analysis revealed that roughly half of the protein expression changes that occur in response to APC loss are independent of &#946-catenin. By combining these two analyses, we identified Misshapen-like kinase 1 (MINK1) as a putative substrate of an alternative APC-containing destruction complex and provide evidence for the potential contribution of MINK1 to APC mutant phenotypes. Collectively, our results highlight the extent and importance of Wntindependent APC functions in epithelial biology and disease.

Accurate methods to assess the pathogenicity of mutations are needed to fully leverage the possibilities of genome sequencing in diagnosis. Current data-driven and bioinformatics approaches are, however, limited by the large number of new variations found in each newly sequenced genome, and often do not provide direct mechanistic insight. Here we demonstrate, for the first time, that saturation mutagenesis, biophysical modeling and co-variation analysis, performed in silico, can predict the abundance, metabolic stability, and function of proteins inside living cells. As a model system, we selected the human mismatch repair protein, MSH2, where missense variants are known to cause the hereditary cancer predisposition disease, known as Lynch syndrome. We show that the majority of disease-causing MSH2 mutations give rise to folding defects and proteasome-dependent degradation rather than inherent loss of function, and accordingly our in silico modeling data accurately identifies disease-causing mutations and outperforms the traditionally used genetic disease predictors. Thus, in conclusion, in silico biophysical modeling should be considered for making genotype-phenotype predictions and for diagnosis of Lynch syndrome, and perhaps other hereditary diseases., Author summary The protein quality control system targets misfolded proteins for degradation. So far it has not been possible from sequence or structural data to predict the biological stability of a misfolded protein, or the effect of mutations on intracellular protein levels. Here we demonstrate that in silico saturation mutagenesis and biophysical calculations of the structural stability of the human mismatch repair protein MSH2 correlate with cellular protein levels, turnover and function. Of 24 different MSH2 variants, some of which are linked to Lynch syndrome, a destabilization of as little as 3 kcal/mol is sufficient to cause rapid degradation via the ubiquitin-proteasome pathway. Thus, biophysical modeling can, to a large extent, predict the metabolic stability of proteins. We also show that the same biophysical calculations can be used to distinguish with high accuracy neutral sequence variation from pathogenic variants, and that the calculations outperform several traditionally used disease predictors. We therefore suggest the method to be of potential value for patient stratification in Lynch syndrome, and perhaps other hereditary diseases.

The ubiquitin–proteasome system (UPS) and macroautophagy (autophagy) are central to normal proteostasis and interdependent in that autophagy is known to compensate for the UPS to alleviate ensuing proteotoxic stress that impairs cell function. UPS and autophagy dysfunctions are believed to have a major role in the pathomechanisms of neurodegenerative disease. Here we show that continued 26S proteasome dysfunction in mouse brain cortical neurons causes paranuclear accumulation of fragmented dysfunctional mitochondria, associated with earlier recruitment of Parkin and lysine 48-linked ubiquitination of mitochondrial outer membrane (MOM) proteins, including Mitofusin-2. Early events also include phosphorylation of p62/SQSTM1 (p62) and increased optineurin, as well as autophagosomal LC3B and removal of some mitochondria, supporting the induction of selective autophagy. Inhibition of the degradation of ubiquitinated MOM proteins with continued 26S proteasome dysfunction at later stages may impede efficient mitophagy. However, continued 26S proteasome dysfunction also decreases the levels of essential autophagy proteins ATG9 and LC3B, which is characterised by decreases in their gene expression, ultimately leading to impaired autophagy. Intriguingly, serine 351 phosphorylation of p62 did not enhance its binding to Keap1 or stabilise the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor in this neuronal context. Nrf2 protein levels were markedly decreased despite transcriptional activation of the Nrf2 gene. Our study reveals novel insights into the interplay between the UPS and autophagy in neurons and is imperative to understanding neurodegenerative disease where long-term proteasome inhibition has been implicated.

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.

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.

The Rho-like GTPase, Rac1, induces cytoskeletal rearrangements required for cell migration. Rac activation is regulated through a number of mechanisms, including control of nucleotide exchange and hydrolysis, regulation of subcellular localization or modulation of protein-expression levels. Here, we identify that the small ubiquitin-like modifier (SUMO) E3-ligase, PIAS3, interacts with Rac1 and is required for increased Rac activation and optimal cell migration in response to hepatocyte growth factor (HGF) signalling. We demonstrate that Rac1 can be conjugated to SUMO-1 in response to hepatocyte growth factor treatment and that SUMOylation is enhanced by PIAS3. Furthermore, we identify non-consensus sites within the polybasic region of Rac1 as the main location for SUMO conjugation. We demonstrate that PIAS3-mediated SUMOylation of Rac1 controls the levels of Rac1-GTP and the ability of Rac1 to stimulate lamellipodia, cell migration and invasion. The finding that a Ras superfamily member can be SUMOylated provides an insight into the regulation of these critical mediators of cell behaviour. Our data reveal a role for SUMO in the regulation of cell migration and invasion.

The post-translational modification of proteins with ubiquitin and ubiquitin-like proteins (Ubl) is vital to many cellular functions, and thus the identification of Ubl targets is key to understanding their function. In most cases, only a small proportion of the cellular pool of proteins is found conjugated to a particular Ubl, making identification of Ubl targets technically challenging. For the purposes of proteomic analyses, we have developed a protocol for the large-scale purification of Ubl-linked proteins that minimizes sample contamination with noncovalent interactors and prevents the cleavage of Ubl-substrate bonds catalyzed by Ubl-specific proteases. This is achieved by introducing a denaturing lysis step (in the presence of sodium dodecyl sulfate and alkylating agents that irreversibly inhibit Ubl proteases) before TAP (tandem affinity purification) that allows for efficient purification of putative Ubl-specific substrates in a form suitable for proteomic analysis. The timescale from cell lysis to purified protein sample is 5-6 d.

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.

International audience; In acute promyelocytic leukaemia (APL), 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 (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. In RNF4-depleted cells, PML protein accumulated 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.

Dynamic modification of proteins with the small ubiquitin-like modifier (SUMO) affects the stability, cellular localization, enzymatic activity, and molecular interactions of a wide spectrum of protein targets. We have developed an in vitro fluorescence-resonance-energy-transfer-based assay that uses bacterially expressed substrates for the rapid and quantitative analysis of SUMO paralog-specific C-terminal hydrolase activity. This assay has applications in SUMO protease characterization, enzyme kinetic analysis, determination of SUMO protease activity in eukaryotic cell extracts, and high-throughput inhibitor screening. In addition, while demonstrating such uses, we show that the SUMO-1 processing activity in crude HeLa cell extracts is far greater than that of SUMO-2, implying that differential maturation rates of SUMO paralogs in vivo may be functionally significant. The high degree of structural conservation across the ubiquitin-like protein superfamily suggests that the general principle of this assay should be applicable to other post-translational protein modification systems.

Promyelocytic leukemia (PML) protein forms the basis of PML nuclear bodies (PML NBs), which control many important processes. We have screened an shRNA library targeting ubiquitin pathway proteins for effects on PML NBs, and identified RNF8 and RNF168 DNA-damage response proteins as negative regulators of PML NBs. Additional studies confirmed that depletion of either RNF8 or RNF168 increased the levels of PML NBs and proteins, whereas overexpression induced loss of PML NBs. RNF168 partially localized to PML NBs through its UMI/MIU1 ubiquitin-interacting region and associated with NBs formed by any PML isoform. The association of RNF168 with PML NBs resulted in increased ubiquitylation and SUMO2 modification of PML. In addition, RNF168 was found to associate with proteins modified by SUMO2 and/or SUMO3 in a manner dependent on its ubiquitin-binding sequences, suggesting that hybrid SUMO-ubiquitin chains can be bound. In vitro assays confirmed that RNF168, preferentially, binds hybrid SUMO2-K63 ubiquitin chains compared with K63-ubiquitin chains or individual SUMO2. Our study identified previously unrecognized roles for RNF8 and RNF168 in the regulation of PML, and a so far unknown preference of RNF168 for hybrid SUMO-ubiquitin chains.

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 beta-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.

The small ubiquitin-like modifier (SUMO) is covalently attached to lysine residues in target proteins and in doing so changes the properties of the modified protein. Here we examine the role of SUMO modification in transcriptional regulation. SUMO addition to components of the transcriptional apparatus does not have a common consequence as it can both activate and repress transcription. In most cases, however, SUMO modification of transcription factors leads to repression and various models to explain this, ranging from retention in nuclear bodies to recruitment of histone deacetylases are discussed.

The protein called 'small ubiquitin-like modifier' (SUMO) is post-translationally linked to target proteins at the varepsilon-amino group of lysine residues. This 'SUMOylation' alters the behavior of the target protein, a change that is utilized to regulate diverse cellular processes. Understanding the target-specific consequences of SUMO modification requires knowledge of the location of conjugation sites, and we have developed a straightforward protocol for the proteome-wide identification of SUMO modification sites using mass spectrometry (MS). The approach described herein requires the expression of a mutant form of SUMO, in which the residue preceding the C-terminal Gly-Gly (diGly) is replaced with a lysine (SUMO(KGG)). Digestion of SUMO(KGG) protein conjugates with endoproteinase Lys-C yields a diGly motif attached to target lysines. Peptides containing this adduct are enriched using a diGly-Lys (K-varepsilon-GG)-specific antibody and identified by MS. This diGly signature is characteristic of SUMO(KGG) conjugation alone, as no other ubiquitin-like protein (Ubl) yields this adduct upon Lys-C digestion. We have demonstrated the utility of the approach in SUMOylation studies, but, in principle, it may be adapted for the site-specific identification of proteins modified by any Ubl. Starting from cell lysis, this protocol can be completed in approximately 5 d.

Human Ubc9 is homologous to ubiquitin-conjugating enzymes. However, instead of conjugating ubiquitin, it conjugates a ubiquitin homologue, small ubiquitin-like modifier 1 (SUMO-1), also known as UBL1, GMP1, SMTP3, PIC1, and sentrin. The SUMO-1 conjugation pathway is very similar to that of ubiquitin with regard to the primary sequences of the ubiquitin-activating enzymes (E1), the three-dimensional structures of the ubiquitin-conjugating enzymes (E2), and the chemistry of the overall conjugation pathway. The interaction of substrates with Ubc9 has been studied using NMR spectroscopy. Peptides with sequences that correspond to those of the SUMO-1 conjugation sites from p53 and c-Jun both bind to a surface adjacent to the active site Cys93 of human Ubc9, which has been previously shown to include residues that demonstrate the most significant dynamics on the microsecond to millisecond time scale. Mutations in this region, Q126A, Q130A, A131D, E132A, Y134A, and T135A, were constructed to evaluate the role of these residues in SUMO-1 conjugation. These alterations have significant effects on the conjugation of SUMO-1 with the target proteins p53, E1B, and promyelocytic leukemia protein and define a substrate binding site on Ubc9. Furthermore, the SUMO-1 conjugation site of p53 does not form any defined secondary structure when either free or bound to Ubc9. This suggests that a defined secondary structure at SUMO-1 conjugation sites in target proteins is not necessary for recognition and conjugation by the SUMO-1 pathway.

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 Thr(90) mutated to Lys. Endoproteinase cleavage with Lys-C of 6His-SUMO2(T90K)-modified proteins from human cell lysates produced a diGly remnant on SUMO2(T90K)-conjugated lysines, enabling immunoprecipitation of SUMO2(T90K)-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.

Summary Dimeric RING E3 ligases interact with protein substrates and conformationally restrain the ubiquitin-E2-conjugating enzyme thioester complex such that it is primed for catalysis. RNF4 is an E3 ligase containing an N-terminal domain that binds its polySUMO substrates and a C-terminal RING domain responsible for dimerization. To investigate how RNF4 activity is controlled, we increased polySUMO substrate concentration by ablating expression of SUMO protease SENP6. Accumulation of SUMO chains in vivo leads to ubiquitin-mediated proteolysis of RNF4. In vitro we demonstrate that at concentrations equivalent to those found in vivo RNF4 is predominantly monomeric and inactive as an ubiquitin E3 ligase. However, in the presence of SUMO chains, RNF4 is activated by dimerization, leading to both substrate ubiquitylation and autoubiquitylation, responsible for degradation of RNF4. Thus the ubiquitin E3 ligase activity of RNF4 is directly linked to the availability of its polySUMO substrates., Graphical Abstract, Highlights • Characterization of the mechanism of activation of the ubiquitin E3 ligase RNF4 • First indication of E3 ligase activation by SUMO chains, RNF4 is an E3 ligase containing an N-terminal domain that binds its polySUMO substrates and a C-terminal RING domain responsible for dimerization. Rojas-Fernandez et al. show that in the presence of SUMO chains RNF4 is activated by dimerization, leading to both substrate ubiquitylation and autoubiquitylation.

The covalent attachment of the protein ubiquitin to intracellular proteins by a process known as ubiquitylation regulates almost all major cellular systems, predominantly by regulating protein turnover. Ubiquitylation requires the co-ordinated action of three enzymes termed E1, E2 and E3, and typically results in the formation of an isopeptide bond between the C-terminal carboxy group of ubiquitin and the ϵ-amino group of a target lysine residue. However, ubiquitin is also known to conjugate to the thiol of cysteine residue side chains and the α-amino group of protein N-termini, although the enzymes responsible for discrimination between different chemical groups have not been defined. In the present study, we show that Ube2W (Ubc16) is an E2 ubiquitin-conjugating enzyme with specific protein N-terminal mono-ubiquitylation activity. Ube2W conjugates ubiquitin not only to its own N-terminus, but also to that of the small ubiquitin-like modifier SUMO (small ubiquitin-related modifier) in a manner dependent on the SUMO-targeted ubiquitin ligase RNF4 (RING finger protein 4). Furthermore, N-terminal mono-ubiquitylation of SUMO-2 primes it for poly-ubiquitylation by the Ubc13–UEV1 (ubiquitin-conjugating enzyme E2 variant 1) heterodimer, showing that N-terminal ubiquitylation regulates protein fate. The description in the present study is the first of an E2-conjugating enzyme with N-terminal ubiquitylation activity, and highlights the importance of E2 enzymes in the ultimate outcome of E3-mediated ubiquitylation.

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.

In humans cells three SUMO paralogues (SUMO-1, SUMO-2 and SUMO-3) and six SUMO specific proteases (SENP1-SENP3 and SENP5-SENP7) are expressed. Together the SUMO proteases perform three distinct functions. They: (1) process the immature pro-SUMO proteins into the active forms, (2) remove SUMO molecules conjugated to protein targets, and (3) depolymerise SUMO conjugated within polymeric chains. By regulating these processes the SENPs play a crucial role in regulating the sumoylation state of target proteins in cells, and therefore are academically and pharmacologically interesting enzymes. Gel-based techniques for SENP analysis are well established and can be used for many applications, but their laborious methodology makes them cumbersome tools for kinetic analysis or inhibitor screening. Therefore in vitro FRET-based assays have been developed to test the three major functions of the SENPs. These use fluorescent protein fusions of the SUMOs, and together facilitate high-throughput, real-time analysis of the three major SUMO protease activities.

In humans cells three SUMO paralogues (SUMO-1, SUMO-2 and SUMO-3) and six SUMO specific proteases (SENP1-SENP3 and SENP5-SENP7) are expressed. Together the SUMO proteases perform three distinct functions. They: (1) process the immature pro-SUMO proteins into the active forms, (2) remove SUMO molecules conjugated to protein targets, and (3) depolymerise SUMO conjugated within polymeric chains. By regulating these processes the SENPs play a crucial role in regulating the sumoylation state of target proteins in cells, and therefore are academically and pharmacologically interesting enzymes. Gel-based techniques for SENP analysis are well established and can be used for many applications, but their laborious methodology makes them cumbersome tools for kinetic analysis or inhibitor screening. Therefore in vitro FRET-based assays have been developed to test the three major functions of the SENPs. These use fluorescent protein fusions of the SUMOs, and together facilitate high-throughput, real-time analysis of the three major SUMO protease activities.

Protein-protein binding and signaling pathways are important fields of biomedical science. Here we report simple optical methods for the determination of the equilibrium binding constant K(d) of protein-protein interactions as well as quantitative studies of biochemical cascades. The techniques are based on steady-state and time-resolved fluorescence resonance energy transfer (FRET) between ECFP and Venus-YFP fused to proteins of the SUMO family. Using FRET has several advantages over conventional free-solution techniques such as isothermal titration calorimetry (ITC): Concentrations are determined accurately by absorbance, highly sensitive binding signals enable the analysis of small quantities, and assays are compatible with multi-well plate format. Most importantly, our FRET-based techniques enable us to measure the effect of other molecules on the binding of two proteins of interest, which is not straightforward with other approaches. These assays provide powerful tools for the study of competitive biochemical cascades and the extent to which drug candidates modify protein interactions.

SUMO1, SUMO2 and SUMO3 are important intracellular proteins that are found attached covalently to, and consequently regulate the function of, many key proteins. In this section, disease links that meet the TPdb Target Validation criteria for the individual SUMO proteins are discussed. Please note that sumoylation of a protein involved in disease and/or changes in protein sumoylation do not in themselves meet the criteria for inclusion. Diseases associated with changes in SUMO modification are discussed in the section Function and Localization: In disease.

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.

Small ubiquitin-like modifier (SUMO)-specific protease SENP1 processes SUMO-1, SUMO-2 and SUMO-3 to mature forms and deconjugates them from modified proteins. To establish the proteolytic mechanism, we determined structures of catalytically inactive SENP1 bound to SUMO-1-modified RanGAP1 and to unprocessed SUMO-1. In each case, the scissile peptide bond is kinked at a right angle to the C-terminal tail of SUMO-1 and has the cis configuration of the amide nitrogens. SENP1 preferentially processes SUMO-1 over SUMO-2, but binding thermodynamics of full-length SUMO-1 and SUMO-2 to SENP1 and K(m) values for processing are very similar. However, k(cat) values differ by 50-fold. Thus, discrimination between unprocessed SUMO-1 and SUMO-2 by SENP1 is based on a catalytic step rather than substrate binding and is likely to reflect differences in the ability of SENP1 to correctly orientate the scissile bonds in SUMO-1 and SUMO-2.

The attachment of the ubiquitin-like protein SUMO to target proteins is involved in a number of important cellular processes. Typically, SUMO modification occurs on lysine residues within the consensus sequence psiKxE/D (psi is a hydrophobic residue and x is any residue), although there are examples of modifications at nonconsensus sites. In most cases, sites of SUMO modification have been inferred from a combination of site-directed mutagenesis and functional analysis; however, these methods have two limitations. They do not directly identify the acceptor lysine, nor are they sufficient to identify acceptor lysine residues in SUMO polymers. Here, we use Fourier transform ion cyclotron resonance (FT-ICR) together with activated-ion electron capture dissociation (AI-ECD) or infrared multiphoton dissociation (IRMPD) mass spectrometry techniques to overcome these restrictions. These approaches were employed to analyze the autoSUMOylation reaction catalyzed by the SUMO E3 ligase RanBP2. Six sites of in vitro SUMOylation in RanBP2 along with four branch-point lysines in SUMO-1 and three in SUMO-2 were identified. In all but one case, SUMOylation occurred within the sequences KxE or KpsiK. These results demonstrate the utility of FT-ICR with AI-ECD or IRMPD mass spectrometry in detecting SUMOylation, and sites of SUMOylation, and their potential roles as complementary tools for proteomic and functional analysis, and provide significant insight into the modification of a SUMO ligase for which conventional techniques have been unsuccessful.

Covalent posttranslational modification of target proteins with ubiquitin and ubiquitin-like proteins regulates many important cellular processes. However, the molecular mechanisms by which these proteins are activated and conjugated to substrates has yet to be fully understood. NMR studies have shown that the ubiquitin-like proteins SUMO-1, -2, and -3 interact with the same N-terminal region of the E2 conjugating enzyme Ubc9 with similar affinities. This is correlated to their almost identical utilization by Ubc9 in the SUMO conjugation pathway. To investigate the functional significance of this interaction, site-directed mutagenesis was used to alter residues in the SUMO binding surface of Ubc9, and the effect of the amino acid substitutions on binding and conjugation to SUMO-1 and target protein RanGAP1 was investigated by isothermal titration calorimetry and biochemical analysis. R13A/K14A and R17A/K18A mutations in Ubc9 disrupted the interaction with SUMO-1 but did not completely abolish the interaction with E1. While these Ubc9 mutants displayed a significantly reduced efficiency in the transfer of SUMO-1 from E1 to E2, their ability to recognize substrate and transfer SUMO-1 from E2 to the target protein was unaffected. These results suggest that the noncovalent binding site of SUMO-1 on Ubc9, although distant from the active site, is important for the transfer of SUMO-1 from the E1 to the E2. The conservation of E2 enzymes across the ubiquitin and ubiquitin-like protein pathways indicates that analogous N-terminal sites of E2 enzymes are likely to have similar roles in general.

The small ubiquitin-like modifier SUMO-1 is covalently attached to lysine residues on target proteins by a specific conjugation pathway involving the El enzyme SAE1/SAE2 and the E2 enzyme Ubc9. In an ATP-dependent manner,the C-terminus of SUMO-1 forms consecutive thiolester bonds with cysteine residues in the SAE2 subunit and Ubc9, before the Ubc9.SUMO-I1thiolester complex catalyzes the formation of an isopeptide bond between SUMO-1 1 and the ∈-amino group of the target lysine residue on the protein substrate. The SUMO- 1 conjugation pathway bears many similarities with that of ubiquitin and other ubiquitin-like protein modifiers (Ub1s), and because of its production of a singly conjugated substrate and the lack of absolute requirement in vitro for E3 enzymes, the SUMO-1/Ubc9 system is a good model for the analysis of protein conjugation pathways that share this basic chemistry. Here we describe methods of both steady-state and half-reaction kinetic analysis of Ubc9, and use these techniques to determine the role of two residues, Asp 1 0 0 and Lys 1 0 1 of Ubc9 which are not found in E2 enzymes from other protein conjugation pathways. These residues are found close to the active site Cys in the tertiary structure of Ubc9, and although they are shown to inhibit the transesterification reaction from SAE1/SAE2, they are important for substrate recognition in the context of the thiolester complex with SUMO-1.

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 psiKXE, 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 detected in 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.