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.

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.

The Sumo proteome of proliferating and neuronal-differentiating cells reveals Utf1 among key Sumo targets involved in neurogenesis.

Post-translational modification by covalent attachment of the Small ubiquitin-like modifier (Sumo) polypeptide regulates a multitude of processes in vertebrates. Despite demonstrated roles of Sumo in the development and function of the nervous system, the identification of key factors displaying a sumoylation-dependent activity during neurogenesis remains elusive. Through a SILAC (stable isotope labeling by/with amino acids in cell culture)-based proteomic approach, we have identified the Sumo proteome of the model cell line P19 under proliferation and neuronal differentiation conditions. More than 300 proteins were identified as putative Sumo targets differentially associated with one or the other condition. A group of proteins of interest were validated and investigated in functional studies. Among these, Utf1 was revealed as a new Sumo target. Gain-of-function experiments demonstrated marked differences between the effects on neurogenesis of overexpressing wild-type and sumoylation mutant versions of the selected proteins. While sumoylation of Prox1, Sall4a, Trim24, and Utf1 was associated with a positive effect on neurogenesis in P19 cells, sumoylation of Kctd15 was associated with a negative effect. Prox1, Sall4a, and Kctd15 were further analyzed in the vertebrate neural tube of living embryos, with similar results. Finally, a detailed analysis of Utf1 showed the sumoylation dependence of Utf1 function in controlling the expression of bivalent genes. Interestingly, this effect seems to rely on two mechanisms: sumoylation modulates binding of Utf1 to the chromatin and mediates recruitment of the messenger RNA-decapping enzyme Dcp1a through a conserved SIM (Sumo-interacting motif). Altogether, our results indicate that the combined sumoylation status of key proteins determines the proper progress of neurogenesis.

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.

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.

The ubiquitin-like modifier FAT10 interferes with SUMO activation.

The covalent attachment of the cytokine-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) to hundreds of substrate proteins leads to their rapid degradation by the 26 S proteasome independently of ubiquitylation. Here, we identify another function of FAT10, showing that it interferes with the activation of SUMO1/2/3 in vitro and down-regulates SUMO conjugation and the SUMO-dependent formation of promyelocytic leukemia protein (PML) bodies in cells. Mechanistically, we show that FAT10 directly binds to and impedes the activity of the heterodimeric SUMO E1 activating enzyme AOS1/UBA2 by competing very efficiently with SUMO for activation and thioester formation. Nevertheless, activation of FAT10 by AOS1/UBA2 does not lead to covalent conjugation of FAT10 with substrate proteins which relies on its cognate E1 enzyme UBA6. Hence, we report that one ubiquitin-like modifier (FAT10) inhibits the conjugation and function of another ubiquitin-like modifier (SUMO) by impairing its activation.

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.

Pyrophosphate modulates plant stress responses via SUMOylation.

Pyrophosphate (PPi), a byproduct of macromolecule biosynthesis is maintained at low levels by soluble inorganic pyrophosphatases (sPPase) found in all eukaryotes. In plants, H+-pumping pyrophosphatases (H+-PPase) convert the substantial energy present in PPi into an electrochemical gradient. We show here, that both cold- and heat stress sensitivity of fugu5 mutants lacking the major H+-PPase isoform AVP1 is correlated with reduced SUMOylation. In addition, we show that increased PPi concentrations interfere with SUMOylation in yeast and we provide evidence that SUMO activating E1-enzymes are inhibited by micromolar concentrations of PPi in a non-competitive manner. Taken together, our results do not only provide a mechanistic explanation for the beneficial effects of AVP1 overexpression in plants but they also highlight PPi as an important integrator of metabolism and stress tolerance.

Control of SUMO and Ubiquitin by ROS: Signaling and disease implications.

Reversible post-translational modifications (PTMs) ensure rapid signal transmission from sensors to effectors. Reversible modification of proteins by the small proteins Ubiquitin and SUMO are involved in virtually all cellular processes and can modify thousands of proteins. Ubiquitination or SUMOylation is the reversible attachment of these modifiers to lysine residues of a target via isopeptide bond formation. These modifications require ATP and an enzymatic cascade composed of three classes of proteins: E1 activating enzymes, E2 conjugating enzymes and E3 ligases. The reversibility of the modification is ensured by specific isopeptidases. E1 and E2 enzymes, some E3 ligases and most isopeptidases have catalytic cysteine residues, which make them potentially susceptible for oxidation. Indeed, an increasing number of examples reveal regulation of ubiquitination and SUMOylation by reactive oxygen species, both in the context of redox signaling and in severe oxidative stress. Importantly, ubiquitination and SUMOylation play essential roles in the regulation of ROS homeostasis, participating in the control of ROS production and clearance. In this review, we will discuss the interplay between ROS homeostasis, Ubiquitin and SUMO pathways and the implications for the oxidative stress response and cell signaling.

IRAK2 directs stimulus-dependent nuclear export of inflammatory mRNAs.

Expression of inflammatory genes is determined in part by post-transcriptional regulation of mRNA metabolism but how stimulus- and transcript-dependent nuclear export influence is poorly understood. Here, we report a novel pathway in which LPS/TLR4 engagement promotes nuclear localization of IRAK2 to facilitate nuclear export of a specific subset of inflammation-related mRNAs for translation in murine macrophages. IRAK2 kinase activity is required for LPS-induced RanBP2-mediated IRAK2 sumoylation and subsequent nuclear translocation. Array analysis showed that an SRSF1-binding motif is enriched in mRNAs dependent on IRAK2 for nuclear export. Nuclear IRAK2 phosphorylates SRSF1 to reduce its binding to target mRNAs, which promotes the RNA binding of the nuclear export adaptor ALYREF and nuclear export receptor Nxf1 loading for the export of the mRNAs. In summary, LPS activates a nuclear function of IRAK2 that facilitates the assembly of nuclear export machinery to export selected inflammatory mRNAs to the cytoplasm for translation.

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.

Reconstitution of the Recombinant RanBP2 SUMO E3 Ligase Complex.

One of the few proteins that have SUMO E3 ligase activity is the 358 kDa nucleoporin RanBP2 (Nup358). While small fragments of RanBP2 can stimulate SUMOylation in vitro, the physiologically relevant E3 ligase is a stable multi-subunit complex comprised of RanBP2, SUMOylated RanGAP1, and Ubc9. Here, we provide a detailed protocol to in vitro reconstitute the RanBP2 SUMO E3 ligase complex. With the exception of RanBP2, reconstitution involves untagged full-length proteins. We describe the bacterial expression and purification of all complex components, namely an 86 kDa His-tagged RanBP2 fragment, the SUMO E2-conjugating enzyme Ubc9, RanGAP1, and SUMO1, and we provide a protocol for quantitative SUMOylation of RanGAP1. Finally, we present details for the assembly and final purification of the catalytically active RanBP2/RanGAP1*SUMO1/Ubc9 complex.

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 SUMO E3 ligase is a disassembly machine for Crm1-dependent nuclear export complexes.

Continuous cycles of nucleocytoplasmic transport require disassembly of transport receptor/Ran-GTP complexes in the cytoplasm. A basic disassembly mechanism in all eukaryotes depends on soluble RanGAP and RanBP1. In vertebrates, a significant fraction of RanGAP1 stably interacts with the nucleoporin RanBP2 at a binding site that is flanked by FG-repeats and Ran-binding domains, and overlaps with RanBP2's SUMO E3 ligase region. Here, we show that the RanBP2/RanGAP1*SUMO1/Ubc9 complex functions as an autonomous disassembly machine with a preference for the export receptor Crm1. We describe three in vitro reconstituted disassembly intermediates, which show binding of a Crm1 export complex via two FG-repeat patches, cargo-release by RanBP2's Ran-binding domains and retention of free Crm1 at RanBP2 after Ran-GTP hydrolysis. Intriguingly, all intermediates are compatible with SUMO E3 ligase activity, suggesting that the RanBP2/RanGAP1*SUMO1/Ubc9 complex may link Crm1- and SUMO-dependent functions.

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.

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.

SUMOylation-dependent LRH-1/PROX1 interaction promotes atherosclerosis by decreasing hepatic reverse cholesterol transport.

Reverse cholesterol transport (RCT) is an antiatherogenic process in which excessive cholesterol from peripheral tissues is transported to the liver and finally excreted from the body via the bile. The nuclear receptor liver receptor homolog 1 (LRH-1) drives expression of genes regulating RCT, and its activity can be modified by different posttranslational modifications. Here, we show that atherosclerosis-prone mice carrying a mutation that abolishes SUMOylation of LRH-1 on K289R develop less aortic plaques than control littermates when exposed to a high-cholesterol diet. The mechanism underlying this atheroprotection involves an increase in RCT and its associated hepatic genes and is secondary to a compromised interaction of LRH-1 K289R with the corepressor prospero homeobox protein 1 (PROX1). Our study reveals that the SUMOylation status of a single nuclear receptor lysine residue can impact the development of a complex metabolic disease such as atherosclerosis.

The Ran GTPase-activating protein (RanGAP1) is critically involved in smooth muscle cell differentiation, proliferation and migration following vascular injury: implications for neointima formation and restenosis.

Differentiation and dedifferentiation, accompanied by proliferation play a pivotal role for the phenotypic development of vascular proliferative diseases (VPD), such as restenosis. Increasing evidence points to an essential role of regulated nucleoporin expression in the choice between differentiation and proliferation. However, whether components of the Ran GTPase cycle, which is of pivotal importance for both nucleocytoplasmic transport and for mitotic progression, are subject to similar regulation in VPD is currently unknown. Here, we show that differentiation of human coronary artery smooth muscle cell (CASMC) to a contractile phenotype by stepwise serum depletion leads to significant reduction of RanGAP1 protein levels. The inverse event, dedifferentiation of cells, was assessed in the rat carotid artery balloon injury model, a well-accepted model for neointima formation and restenosis. As revealed by temporospatial analysis of RanGAP1 expression, neointima formation in rat carotid arteries was associated with a significant upregulation of RanGAP1 expression at 3 and 7 days after balloon injury. Of note, neointimal cells located at the luminal surface revealed persistent RanGAP1 expression, as opposed to cells in deeper layers of the neointima where RanGAP1 expression was less or not detectable at all. To gain first evidence for a direct influence of RanGAP1 levels on differentiation, we reduced RanGAP1 in human coronary artery smooth muscle cells by siRNA. Indeed, downregulation of the essential RanGAP1 protein by 50% induced a differentiated, spindle-like smooth muscle cell phenotype, accompanied by an upregulation of the differentiation marker desmin. Reduction of RanGAP1 levels also resulted in a reduction of mitogen induced cellular migration and proliferation as well as a significant upregulation of the cyclin-dependent kinase inhibitor p27KIP1, without evidence for cellular necrosis. These findings suggest that RanGAP1 plays a critical role in smooth muscle cell differentiation, migration and proliferation in vitro and in vivo. Appropriate modulation of RanGAP1 expression may thus be a strategy to modulate VPD development such as restenosis.

Identification and analysis of endogenous SUMO1 and SUMO2/3 targets in mammalian cells and tissues using monoclonal antibodies.

SUMOylation is a protein modification that regulates the function of hundreds of proteins. Detecting endogenous SUMOylation is challenging: most small ubiquitin-related modifier (SUMO) targets are low in abundance, and only a fraction of a protein's cellular pool is typically SUMOylated. Here we present a step-by-step protocol for the enrichment of endogenous SUMO targets from mammalian cells and tissues (specifically, mouse liver), based on the use of monoclonal antibodies that are available to the scientific community. The protocol comprises (i) production of antibodies and affinity matrix, (ii) denaturing cell lysis, and (iii) SUMO immunoprecipitation followed by peptide elution. Production of affinity matrix and cell lysis requires ∼1 d. The immunoprecipitation with peptide elution can be performed in 2 d. As SUMO proteins are conserved, this protocol should also be applicable to other organisms, including many vertebrates and Drosophila melanogaster.