Sumoylation: a regulatory protein modification in health and disease.

Posttranslational modification with small ubiquitin-related modifier (SUMO) proteins is now established as one of the key regulatory protein modifications in eukaryotic cells. Hundreds of proteins involved in processes such as chromatin organization, transcription, DNA repair, macromolecular assembly, protein homeostasis, trafficking, and signal transduction are subject to reversible sumoylation. Hence, it is not surprising that disease links are beginning to emerge and that interference with sumoylation is being considered for intervention. Here, we summarize basic mechanisms and highlight recent developments in the physiology of sumoylation.

SCFFbxw5 targets kinesin-13 proteins to facilitate ciliogenesis.

Microtubule depolymerases of the kinesin-13 family play important roles in various cellular processes and are frequently overexpressed in different cancer types. Despite the importance of their correct abundance, remarkably little is known about how their levels are regulated in cells. Using comprehensive screening on protein microarrays, we identified 161 candidate substrates of the multi-subunit ubiquitin E3 ligase SCFFbxw5 , including the kinesin-13 member Kif2c/MCAK. In vitro reconstitution assays demonstrate that MCAK and its closely related orthologs Kif2a and Kif2b become efficiently polyubiquitylated by neddylated SCFFbxw5 and Cdc34, without requiring preceding modifications. In cells, SCFFbxw5  targets MCAK for proteasomal degradation predominantly during G2 . While this seems largely dispensable for mitotic progression, loss of Fbxw5 leads to increased MCAK levels at basal bodies and impairs ciliogenesis in the following G1 /G0 , which can be rescued by concomitant knockdown of MCAK, Kif2a or Kif2b. We thus propose a novel regulatory event of ciliogenesis that begins already within the G2 phase of the preceding cell cycle.

Fasting-sensitive SUMO-switch on Prox1 controls hepatic cholesterol metabolism.

Accumulation of excess nutrients hampers proper liver function and is linked to nonalcoholic fatty liver disease (NAFLD) in obesity. However, the signals responsible for an impaired adaptation of hepatocytes to obesogenic dietary cues remain still largely unknown. Post-translational modification by the small ubiquitin-like modifier (SUMO) allows for a dynamic regulation of numerous processes including transcriptional reprogramming. We demonstrate that specific SUMOylation of transcription factor Prox1 represents a nutrient-sensitive determinant of hepatic fasting metabolism. Prox1 is highly SUMOylated on lysine 556 in the liver of ad libitum and refed mice, while this modification is abolished upon fasting. In the context of diet-induced obesity, Prox1 SUMOylation becomes less sensitive to fasting cues. The hepatocyte-selective knock-in of a SUMOylation-deficient Prox1 mutant into mice fed a high-fat/high-fructose diet leads to a reduction of systemic cholesterol levels, associated with the induction of liver bile acid detoxifying pathways during fasting. The generation of tools to maintain the nutrient-sensitive SUMO-switch on Prox1 may thus contribute to the development of "fasting-based" approaches for the preservation of metabolic health.

Transient deSUMOylation of IRF2BP proteins controls early transcription in EGFR signaling.

Molecular switches are essential modules in signaling networks and transcriptional reprogramming. Here, we describe a role for small ubiquitin-related modifier SUMO as a molecular switch in epidermal growth factor receptor (EGFR) signaling. Using quantitative mass spectrometry, we compare the endogenous SUMO proteomes of HeLa cells before and after EGF stimulation. Thereby, we identify a small group of transcriptional coregulators including IRF2BP1, IRF2BP2, and IRF2BPL as novel players in EGFR signaling. Comparison of cells expressing wild type or SUMOylation-deficient IRF2BP1 indicates that transient deSUMOylation of IRF2BP proteins is important for appropriate expression of immediate early genes including dual specificity phosphatase 1 (DUSP1, MKP-1) and the transcription factor ATF3. We find that IRF2BP1 is a repressor, whose transient deSUMOylation on the DUSP1 promoter allows-and whose timely reSUMOylation restricts-DUSP1 transcription. Our work thus provides a paradigm how comparative SUMO proteome analyses serve to reveal novel regulators in signal transduction and transcription.

Heat shock transcription factor 1 is SUMOylated in the activated trimeric state.

The heat shock response is a transcriptional program of organisms to counteract an imbalance in protein homeostasis. It is orchestrated in all eukaryotic cells by heat shock transcription factor 1 (Hsf1). Despite very intensive research, the intricacies of the Hsf1 activation-attenuation cycle remain elusive at a molecular level. Post-translational modifications belong to one of the key mechanisms proposed to adapt the Hsf1 activity to the needs of individual cells, and phosphorylation of Hsf1 at multiple sites has attracted much attention. According to cell biological and proteomics data, Hsf1 is also modified by small ubiquitin-like modifier (SUMO) at several sites. How SUMOylation affects Hsf1 activity at a molecular level is still unclear. Here, we analyzed Hsf1 SUMOylation in vitro with purified components to address questions that could not be answered in cell culture models. In vitro Hsf1 is primarily conjugated at lysine 298 with a single SUMO, though we did detect low-level SUMOylation at other sites. Different SUMO E3 ligases such as protein inhibitor of activated STAT 4 enhanced the efficiency of in vitro modification but did not alter SUMO site preferences. We provide evidence that Hsf1 trimerization and phosphorylation at serines 303 and 307 increases SUMOylation efficiency, suggesting that Hsf1 is SUMOylated in its activated state. Hsf1 can be SUMOylated when DNA bound, and SUMOylation of Hsf1 does neither alter DNA-binding affinity nor affects heat shock cognate 71kDa protein (HSPA8)+DnaJ homolog subfamily B member 1-mediated monomerization of Hsf1 trimers and concomitant dislocation from DNA. We propose that SUMOylation acts at the transcription level of the heat shock response.

Hypoxia-induced Changes in SUMO Conjugation Affect Transcriptional Regulation Under Low Oxygen.

Hypoxia occurs in pathological conditions, such as cancer, as a result of the imbalance between oxygen supply and consumption by proliferating cells. HIFs are critical molecular mediators of the physiological response to hypoxia but also regulate multiple steps of carcinogenesis including tumor progression and metastasis. Recent data support that sumoylation, the covalent attachment of the Small Ubiquitin-related MOdifier (SUMO) to proteins, is involved in the activation of the hypoxic response and the ensuing signaling cascade. To gain insights into differences of the SUMO1 and SUMO2/3 proteome of HeLa cells under normoxia and cells grown for 48 h under hypoxic conditions, we employed endogenous SUMO-immunoprecipitation in combination with quantitative mass spectrometry (SILAC). The group of proteins whose abundance was increased both in the total proteome and in the SUMO IPs from hypoxic conditions was enriched in enzymes linked to the hypoxic response. In contrast, proteins whose SUMOylation status changed without concomitant change in abundance were predominantly transcriptions factors or transcription regulators. Particularly interesting was transcription factor TFAP2A (Activating enhancer binding Protein 2 alpha), whose sumoylation decreased on hypoxia. TFAP2A is known to interact with HIF-1 and we provide evidence that deSUMOylation of TFAP2A enhances the transcriptional activity of HIF-1 under hypoxic conditions. Overall, these results support the notion that SUMO-regulated signaling pathways contribute at many distinct levels to the cellular response to low oxygen.

Redox regulation of SUMO enzymes is required for ATM activity and survival in oxidative stress.

To sense and defend against oxidative stress, cells depend on signal transduction cascades involving redox-sensitive proteins. We previously identified SUMO (small ubiquitin-related modifier) enzymes as downstream effectors of reactive oxygen species (ROS). Hydrogen peroxide transiently inactivates SUMO E1 and E2 enzymes by inducing a disulfide bond between their catalytic cysteines. How important their oxidation is in light of many other redox-regulated proteins has however been unclear. To selectively disrupt this redox switch, we identified a catalytically fully active SUMO E2 enzyme variant (Ubc9 D100A) with strongly reduced propensity to maintain a disulfide with the E1 enzyme in vitro and in cells. Replacement of Ubc9 by this variant impairs cell survival both under acute and mild chronic oxidative stresses. Intriguingly, Ubc9 D100A cells fail to maintain activity of the ATM-Chk2 DNA damage response pathway that is induced by hydrogen peroxide. In line with this, these cells are also more sensitive to the ROS-producing chemotherapeutic drugs etoposide/Vp16 and Ara-C. These findings reveal that SUMO E1~E2 oxidation is an essential redox switch in oxidative stress.

The RanBP2/RanGAP1*SUMO1/Ubc9 complex is a multisubunit SUMO E3 ligase.

RanBP2/Nup358 is an essential protein with roles in nuclear transport and mitosis, and is one of the few known SUMO E3 ligases. However, why RanBP2 functions in vivo has been unclear: throughout the cell cycle it stably interacts with RanGAP1*SUMO1 and Ubc9, whose binding sites overlap with the E3 ligase region. Here we show that cellular RanBP2 is quantitatively associated with RanGAP1, indicating that complexed rather than free RanBP2 is the relevant E3 ligase. Biochemical reconstitution of the RanBP2/RanGAP1*SUMO1/Ubc9 complex enabled us to characterize its activity on the endogenous substrate Borealin. We find that the complex is a composite E3 ligase rather than an E2-E3 complex, and demonstrate that complex formation induces activation of a catalytic site that shows no activity in free RanBP2. Our findings provide insights into the mechanism of an important E3 ligase, and extend the concept of multisubunit E3 ligases from ubiquitin to the SUMO field.

Thiolutin is a zinc chelator that inhibits the Rpn11 and other JAMM metalloproteases.

Thiolutin is a disulfide-containing antibiotic and anti-angiogenic compound produced by Streptomyces. Its biological targets are not known. We show that reduced thiolutin is a zinc chelator that inhibits the JAB1/MPN/Mov34 (JAMM) domain-containing metalloprotease Rpn11, a deubiquitinating enzyme of the 19S proteasome. Thiolutin also inhibits the JAMM metalloproteases Csn5, the deneddylase of the COP9 signalosome; AMSH, which regulates ubiquitin-dependent sorting of cell-surface receptors; and BRCC36, a K63-specific deubiquitinase of the BRCC36-containing isopeptidase complex and the BRCA1-BRCA2-containing complex. We provide evidence that other dithiolopyrrolones also function as inhibitors of JAMM metalloproteases.

Concepts in sumoylation: a decade on.

A decade has passed since SUMO (small ubiquitin-related modifier) was discovered to be a reversible post-translational protein modifier. During this time many enzymes that participate in regulated SUMO-conjugation and -deconjugation pathways have been identified and characterized. In parallel, the search for SUMO substrates has produced a long list of targets, which appear to be involved in most cellular functions. Sumoylation is a highly dynamic process and its outcomes are extremely diverse, ranging from changes in localization to altered activity and, in some cases, stability of the modified protein. At first glance, these effects have nothing in common; however, it seems that they all result from changes in the molecular interactions of the sumoylated proteins.

Sumoylation of the GTPase Ran by the RanBP2 SUMO E3 Ligase Complex.

The SUMO E3 ligase complex RanBP2/RanGAP1*SUMO1/Ubc9 localizes at cytoplasmic nuclear pore complex (NPC) filaments and is a docking site in nucleocytoplasmic transport. RanBP2 has four Ran binding domains (RBDs), two of which flank RanBP2's E3 ligase region. We thus wondered whether the small GTPase Ran is a target for RanBP2-dependent sumoylation. Indeed, Ran is sumoylated both by a reconstituted and the endogenous RanBP2 complex in semi-permeabilized cells. Generic inhibition of SUMO isopeptidases or depletion of the SUMO isopeptidase SENP1 enhances sumoylation of Ran in semi-permeabilized cells. As Ran is typically associated with transport receptors, we tested the influence of Crm1, Imp ß, Transportin, and NTF2 on Ran sumoylation. Surprisingly, all inhibited Ran sumoylation. Mapping Ran sumoylation sites revealed that transport receptors may simply block access of the E2-conjugating enzyme Ubc9, however the acceptor lysines are perfectly accessible in Ran/NTF2 complexes. Isothermal titration calorimetry revealed that NTF2 prevents sumoylation by reducing RanGDP's affinity to RanBP2's RBDs to undetectable levels. Taken together, our findings indicate that RanGDP and not RanGTP is the physiological target for the RanBP2 SUMO E3 ligase complex. Recognition requires interaction of Ran with RanBP2's RBDs, which is prevented by the transport factor NTF2.

SUMO unloads the Kap114 cab.

Nucleocytoplasmic transport is an essential mechanism in all eukaryotic cells, for which the basic mechanisms seemed well understood. Transport receptors of the importin b/karyopherin family recognize, translocate and discharge cargo. Key to directed transport is the GTPase Ran, which determines compartment-specific interactions between receptors and their cargo. In this issue of The EMBO Journal, Rothenbusch et al (2012) now add a new energy-dependent event to this basic pathway by providing direct evidence that the posttranslational modification of the yeast import receptor Kap114 with small ubiquitin-related modifier (SUMO) is indispensable for its correct function. An exciting model emerges in which Kap114 sumoylation regulates Ran-dependent cargo release, and thereby acts as a mechanism for intranuclear targeting of the import cargo.

A role for the Cajal-body-associated SUMO isopeptidase USPL1 in snRNA transcription mediated by RNA polymerase II.

Cajal bodies are nuclear structures that are involved in biogenesis of snRNPs and snoRNPs, maintenance of telomeres and processing of histone mRNA. Recently, the SUMO isopeptidase USPL1 was identified as a component of Cajal bodies that is essential for cellular growth and Cajal body integrity. However, a cellular function for USPL1 is so far unknown. Here, we use RNAi-mediated knockdown in human cells in combination with biochemical and fluorescence microscopy approaches to investigate the function of USPL1 and its link to Cajal bodies. We demonstrate that levels of snRNAs transcribed by RNA polymerase (RNAP) II are reduced upon knockdown of USPL1 and that downstream processes such as snRNP assembly and pre-mRNA splicing are compromised. Importantly, we find that USPL1 associates directly with U snRNA loci and that it interacts and colocalises with components of the Little Elongation Complex, which is involved in RNAPII-mediated snRNA transcription. Thus, our data indicate that USPL1 plays a key role in RNAPII-mediated snRNA transcription.

Mechanism and consequences for paralog-specific sumoylation of ubiquitin-specific protease 25.

Vertebrates express two distinct families of SUMO proteins (SUMO1 and SUMO2/3) that serve distinct functions as posttranslational modifiers. Many proteins are modified specifically with SUMO1 or SUMO2/3, but the mechanisms for paralog selectivity are poorly understood. In a screen for SUMO2/3 binding proteins, we identified Ubiquitin Specific Protease 25 (USP25). USP25 turned out to also be a target for sumoylation, being more efficient with SUMO2/3. Sumoylation takes place within USP25's two ubiquitin interaction motifs (UIMs) that are required for efficient hydrolysis of ubiquitin chains. USP25 sumoylation impairs binding to and hydrolysis of ubiquitin chains. Both SUMO2/3-specific binding and sumoylation depend on a SUMO interaction motif (SIM/SBM). Seven amino acids in the SIM of USP25 are sufficient for SUMO2/3-specific binding and conjugation, even when taken out of structural context. One mechanism for paralog-specific sumoylation may, thus, involve SIM-dependent recruitment of SUMO1 or SUMO2/3 thioester-charged Ubc9 to targets.

A stable chemical SUMO1-Ubc9 conjugate specifically binds as a thioester mimic to the RanBP2-E3 ligase complex.

Ubiquitin and ubiquitin-like (Ubl) modifiers such as SUMO are conjugated to substrate proteins by E1, E2, and E3 enzymes. In the presence of an E3 ligase, the E2∼Ubl thioester intermediate becomes highly activated and is prone to chemical decomposition, thus making biochemical and structural studies difficult. Here we explored a stable chemical conjugate of the E2 enzyme from the SUMO pathway, Ubc9, with its modifier SUMO1 as a structural analogue of the Ubc9∼SUMO1 thioester intermediate, by introducing a triazole linkage by biorthogonal click chemistry. The chemical conjugate proved stable against proteolytic cleavage, in contrast to a Ubc9-SUMO1 isopeptide analogue obtained by auto-SUMOylation. Triazole-linked Ubc9-SUMO1 bound specifically to the preassembled E3 ligase complex RanBP2/RanGAP1*SUMO1/Ubc9, thus suggesting that it is a suitable thioester mimic. We anticipate interesting prospects for its use as a research tool to study protein complexes involving E2 and E3 enzymes.

In vivo localization and identification of SUMOylated proteins in the brain of His6-HA-SUMO1 knock-in mice.

SUMOylation, an essential posttranslational protein modification, is involved in many eukaryotic cellular signaling pathways. The identification of SUMOylated proteins is difficult, because SUMOylation sites in proteins are hard to predict, SUMOylated protein states are transient in vivo and labile in vitro, only a small substrate fraction is SUMOylated in vivo, and identification tools for natively SUMOylated proteins are rare. To solve these problems, we generated knock-in mice expressing His(6)-HA-SUMO1. By anti-HA immunostaining, we show that SUMO1 conjugates in neurons are only detectable in nuclei and annulate lamellae. By anti-HA affinity purification, we identified several hundred candidate SUMO1 substrates, of which we validated Smchd1, Ctip2, TIF1γ, and Zbtb20 as novel substrates. The knock-in mouse represents an excellent mammalian model for studies on SUMO1 localization and screens for SUMO1 conjugates in vivo.

Ubiquitin-specific protease-like 1 (USPL1) is a SUMO isopeptidase with essential, non-catalytic functions.

Isopeptidases are essential regulators of protein ubiquitination and sumoylation. However, only two families of SUMO isopeptidases are at present known. Here, we report an activity-based search with the suicide inhibitor haemagglutinin (HA)-SUMO-vinylmethylester that led to the identification of a surprising new SUMO protease, ubiquitin-specific protease-like 1 (USPL1). Indeed, USPL1 neither binds nor cleaves ubiquitin, but is a potent SUMO isopeptidase both in vitro and in cells. C13orf22l--an essential but distant zebrafish homologue of USPL1--also acts on SUMO, indicating functional conservation. We have identified invariant USPL1 residues required for SUMO binding and cleavage. USPL1 is a low-abundance protein that colocalizes with coilin in Cajal bodies. Its depletion does not affect global sumoylation, but causes striking coilin mislocalization and impairs cell proliferation, functions that are not dependent on USPL1 catalytic activity. Thus, USPL1 represents a third type of SUMO protease, with essential functions in Cajal body biology.

A novel SUMO1-specific interacting motif in dipeptidyl peptidase 9 (DPP9) that is important for enzymatic regulation.

Sumoylation affects many cellular processes by regulating the interactions of modified targets with downstream effectors. Here we identified the cytosolic dipeptidyl peptidase 9 (DPP9) as a SUMO1 interacting protein. Surprisingly, DPP9 binds to SUMO1 independent of the well known SUMO interacting motif, but instead interacts with a loop involving Glu(67) of SUMO1. Intriguingly, DPP9 selectively associates with SUMO1 and not SUMO2, due to a more positive charge in the SUMO1-loop. We mapped the SUMO-binding site of DPP9 to an extended arm structure, predicted to directly flank the substrate entry site. Importantly, whereas mutants in the SUMO1-binding arm are less active compared with wild-type DPP9, SUMO1 stimulates DPP9 activity. Consistent with this, silencing of SUMO1 leads to a reduced cytosolic prolyl-peptidase activity. Taken together, these results suggest that SUMO1, or more likely, a sumoylated protein, acts as an allosteric regulator of DPP9.

Exploring the association between genetic variation in the SUMO isopeptidase gene USPL1 and breast cancer through integration of data from the population-based GENICA study and external genetic databases.

Small ubiquitin-like modifier (SUMO) proteins are covalently attached to target proteins to modify their function. SUMO conjugation participates in processes tightly linked to tumorigenesis. Recently USPL1 (ubiquitin-specific peptidase-like (1) was identified as a SUMO isopeptidase. We report here on the first exploratory study investigating the relationship between genetic variability in USPL1 and breast cancer. Three potentially functional nonsynonymous coding SNPs (rs3742303, rs17609459, rs7984952) were genotyped in 1,021 breast cancer cases and 1,015 controls from the population-based GENICA study. We took advantage of multiple genotype imputation based on HapMap and the 1000 Genomes Project data to refine the association screening in the investigated region. Public genetic databases were also used to investigate the relationship with USPL1 expression in lymphoblastoid cell lines and breast tissue. Women homozygous for the minor C allele of rs7984952 showed a lower risk of Grade 3 breast tumors compared to TT homozygotes (OR 0.50, 95% CI 0.30-0.81). Case-only analyses confirmed the association between rs7984952 and tumor grade (OR 0.60, 95% CI 0.39-0.93). Imputation results in a 238 kb region around rs7984952 based on HapMap and the 1000 Genomes Project data were similar. No imputed variant showed an association signal stronger than rs7984952. USPL1 expression in tumor breast tissue increased with the number of C alleles. The present study illustrates the contribution of multiple imputation of genotypes using public data repositories to standard genotyping laboratory. The provided information may facilitate the design of independent studies to validate the association between USPL1 rs7984952 and risk of Grade 3 breast tumors.

Bicaudal D2, dynein, and kinesin-1 associate with nuclear pore complexes and regulate centrosome and nuclear positioning during mitotic entry.

BICD2 is one of the two mammalian homologues of the Drosophila Bicaudal D, an evolutionarily conserved adaptor between microtubule motors and their cargo that was previously shown to link vesicles and mRNP complexes to the dynein motor. Here, we identified a G2-specific role for BICD2 in the relative positioning of the nucleus and centrosomes in dividing cells. By combining mass spectrometry, biochemical and cell biological approaches, we show that the nuclear pore complex (NPC) component RanBP2 directly binds to BICD2 and recruits it to NPCs specifically in G2 phase of the cell cycle. BICD2, in turn, recruits dynein-dynactin to NPCs and as such is needed to keep centrosomes closely tethered to the nucleus prior to mitotic entry. When dynein function is suppressed by RNA interference-mediated depletion or antibody microinjection, centrosomes and nuclei are actively pushed apart in late G2 and we show that this is due to the action of kinesin-1. Surprisingly, depletion of BICD2 inhibits both dynein and kinesin-1-dependent movements of the nucleus and cytoplasmic NPCs, demonstrating that BICD2 is needed not only for the dynein function at the nuclear pores but also for the antagonistic activity of kinesin-1. Our study demonstrates that the nucleus is subject to opposing activities of dynein and kinesin-1 motors and that BICD2 contributes to nuclear and centrosomal positioning prior to mitotic entry through regulation of both dynein and kinesin-1.