Small Ubiquitin-like Modifier 1 Modification Research Articles

SUMO1 Modification Stabilizes TET3 Protein and Increases Colorectal Cancer Radiation Therapy Sensitivity

The aim of this work was to explore the role and mechanism of active DNA demethylase in colorectal cancer (CRC) radiation sensitization and better understand the function of DNA demethylation in tumor radiosensitization. Tested the effect of ten-eleven translocation 3 (TET3) overexpression on the sensitivity of CRC to radiation therapy through G2/M arrest, apoptosis, and clonogenic suppression. TET3 knockdown HCT 116 and TET3 knockdown LS 180 cell lines were constructed by siRNA technology, and the effect of exogenous knockdown of TET3 on radiation-induced apoptosis, cell cycle arrest, DNA damage, and clone formation in CRC cells were detected. The co-localization of TET3 and smallubiquitin-like modifier 1 (SUMO1), SUMO2/3 was detected by immunofluorescence and cytoplasmic-nuclear extraction, and the interaction between TET3 and SUMO1, SUMO2/3 was detected by a coimmunoprecipitation assay. The malignant phenotype and radiosensitivity of CRC cell lines were favorably linked with TET3 protein and mRNA expression. TET3 is upregulated in 23 of the 27 tumor types investigated, including colon cancer. TET3 was shown to correlate with the CRC pathologic malignancy grade positively. Overexpression of TET3 in CRC cell lines increased radiation-induced apoptosis, G2/M phase arrest, DNA damage, and clonal suppression in vitro. The binding region of TET3 and SUMO2/3 was located at 833-1795 AA except for K1012, K1188, K1397, and K1623. SUMOylation of TET3 increased the stability of the TET3 protein without changing its nuclear localization. We report the sensitizing role of TET3 protein in the radiation of CRC cells, depending on SUMO1 modification of TET3 at the lysine sites (K479, K758, K1012, K1188, K1397, K1623), in turn stabilizing TET3 expression in the nucleus and subsequently increasing the sensitivity of CRC to radiation therapy. Together, this study highlights the potentially critical role of TET3 SUMOylation in radiation regulation, which may contribute to an enhanced understanding of the relationship between DNA demethylation and radiation therapy.

Deletion of SUMO1 attenuates behavioral and anatomical deficits by regulating autophagic activities in Huntington disease

The CAG expansion of huntingtin (mHTT) associated with Huntington disease (HD) is a ubiquitously expressed gene, yet it prominently damages the striatum and cortex, followed by widespread peripheral defects as the disease progresses. However, the underlying mechanisms of neuronal vulnerability are unclear. Previous studies have shown that SUMO1 (small ubiquitin-like modifier-1) modification of mHtt promotes cellular toxicity, but the invivo role and functions of SUMO1 in HD pathogenesis are unclear. Here, we report that SUMO1 deletion in Q175DN HD-het knockin mice (HD mice) prevented age-dependent HD-like motor and neurological impairments and suppressed the striatal atrophy and inflammatory response. SUMO1 deletion caused a drastic reduction in soluble mHtt levels and nuclear and extracellular mHtt inclusions while increasing cytoplasmic mHtt inclusions in the striatum of HD mice. SUMO1 deletion promoted autophagic activity, characterized by augmented interactions between mHtt inclusions and a lysosomal marker (LAMP1), increased LC3B- and LAMP1 interaction, and decreased interaction of sequestosome-1 (p62) and LAMP1 in DARPP-32-positive medium spiny neurons in HD mice. Depletion of SUMO1 in an HD cell model also diminished the mHtt levels and enhanced autophagy flux. In addition, the SUMOylation inhibitor ginkgolic acid strongly enhanced autophagy and diminished mHTT levels in human HD fibroblasts. These results indicate that SUMO is a critical therapeutic target in HD and that blocking SUMO may ameliorate HD pathogenesis by regulating autophagy activities.

The Molecular Simulation Study of nNOS Activation Induced by the Interaction Between Its Calmodulin-Binding Domain and SUMO1

Neuronal nitric oxide synthase (nNOS), an enzyme required for learning and memory, catalyzes L-arginine decomposition during nitric oxide production in mammalian neurons. Over-activation of nNOS leads to oxidative/nitrosative stress, which is part of the pathophysiological process of various neuropsychiatric disorders. Previous experimental studies suggest that nNOS is a target for small ubiquitin-like modifier 1 (SUMO1), and that SUMO1-ylation upregulates nNOS catalytic activity in hippocampal neurons. To date, a comprehensive structural model has not been proposed for nNOS SUMO1-ylation. In this study, our aim was to build in silico models to identify the non-bonded interactions between SUMO1 and the calmodulin binding domain (CaMBD) of nNOS. Using molecular docking and molecular dynamics simulation, we found that SUMO1 modification stabilizes the conformation of nNOS CaMBD, and helps maintain a conformation beneficial for nNOS catalysis. Analysis of the polar contacts and hydrogen bonds, and the root mean square derivation results showed that R726 and R727 of CaMBD formed polar contacts or high occupancy hydrogen bonds with SUMO1. Correlation factor analysis and free energy calculations showed that the W716, L734, F740, M745, and F781 residues were also involved in the SUMO1/CaMBD interaction in an orientation-dependent manner. The potential inhibitor binding pocket of SUMO1, aimed at disrupting SUMO1/CaMBD binding, was detected from the virtual screening results. Our in silico studies revealed that interfering with the non-bonded interactions of SUMO1/CaMBD would blocked nNOS SUMO-ylation and subsequent hyperactivation. This work provides novel structural insight into the functional regulation of nNOS by post-translational SUMO1 modification, and provides suggestions for the design of drugs targeting nNOS hyperactivation.

SUMO1 modification of PKD2 channels regulates arterial contractility

PKD2 (polycystin-2, TRPP1) channels are expressed in a wide variety of cell types and can regulate functions, including cell division and contraction. Whether posttranslational modification of PKD2 modifies channel properties is unclear. Similarly uncertain are signaling mechanisms that regulate PKD2 channels in arterial smooth muscle cells (myocytes). Here, by studying inducible, cell-specific Pkd2 knockout mice, we discovered that PKD2 channels are modified by SUMO1 (small ubiquitin-like modifier 1) protein in myocytes of resistance-size arteries. At physiological intravascular pressures, PKD2 exists in approximately equal proportions as either nonsumoylated (PKD2) or triple SUMO1-modifed (SUMO-PKD2) proteins. SUMO-PKD2 recycles, whereas unmodified PKD2 is surface-resident. Intravascular pressure activates voltage-dependent Ca2+ influx that stimulates the return of internalized SUMO-PKD2 channels to the plasma membrane. In contrast, a reduction in intravascular pressure, membrane hyperpolarization, or inhibition of Ca2+ influx leads to lysosomal degradation of internalized SUMO-PKD2 protein, which reduces surface channel abundance. Through this sumoylation-dependent mechanism, intravascular pressure regulates the surface density of SUMO-PKD2-mediated Na+ currents (INa) in myocytes to control arterial contractility. We also demonstrate that intravascular pressure activates SUMO-PKD2, not PKD2, channels, as desumoylation leads to loss of INa activation in myocytes and vasodilation. In summary, this study reveals that PKD2 channels undergo posttranslational modification by SUMO1, which enables physiological regulation of their surface abundance and pressure-mediated activation in myocytes and thus control of arterial contractility.

Sumoylation of Pyruvate Kinase M2 Inhibits Myeloid Differentiation in Hematopoietic Cells

The pyruvate kinase (PK) is a rate-limiting glycolytic enzyme catalyzing the dephosphorylation of phosphoenolpyruvate to pyruvate. M2 form of PK (PKM2) is expressed during embryogenesis and is the predominant form in tumors of different types. In contrast to the essential role of PKM2 in solid tumors, much less is known about the effects of PKM2 in hematopoietic cells and the development of leukemia. Here we found that PKM2 is modified by small ubiquitin-like modifier 1(SUMO1), which can be reduced by a SUMO1-specific protease SENP1 in hematopoietic cells. SUMOylation induced nuclear localization and conformation change from tetramer to dimer of PKM2. Importantly, SUMOylation of PKM2 is prevalent in a variety of leukemic cell lines as well as primary samples from patients with hematologic malignancies. In consistency, predominant nuclear localization and dimeric forms of PKM2 in leukemic cells were observed. Using in vitro SUMOylation reaction-coupled liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), we identified K270 lysine residue of PKM2 as the SUMOylation target. Replacement of endogenous PKM2 with mutant PKM2K270 showed a significant shift of PKM2 from tetramer to dimer. To investigate the potential leukemogenic effect of PKM2 SUMOylation, murine hematopoietic progenitor 32D clone 3 (32Dcl3) transfectants expressing wild type(WT) or mutant PKM2K270 were generated and G-CSF-induced differentiation was evaluated by morphology appearance and expression of myeloid associated surface markers CD11b and Gr-1. The results showed that expression of WT PKM2 but not mutant PKM2K270 significantly blocked myeloid differentiation. Further investigations revealed that SUMO1 modification of PKM2 at K270 is essential in mediating the interaction between PKM2 and Runt-related transcription factor 1(RUNX1), a master transcriptional factor implicated in the differentiation of hematopoietic cells. This interaction led to a downregulation of RUNX1 during G-CSF-induced myeloid differentiation of 32D cells, which could be abrogated by expression of mutant PKM2K270. Collectively, these data indicated that SUMOylated PKM2 blocks myeloid differentiation through suppressing RUNX1. These findings reveal a novel nonmetabolic function of PKM2 in modulating myeloid differentiation and highlight the critical role of SUMOylation in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Small ubiquitin-like modifier 1 modification of pyruvate kinase M2 promotes aerobic glycolysis and cell proliferation in A549 human lung cancer cells.

ObjectiveLung cancer is the leading cause of cancer-related death worldwide. Aerobic glycolysis is considered the seventh hallmark of cancer. The M2 isoform of pyruvate kinase (PKM2) is an important rate-limiting enzyme in glycolytic pathway, and is strongly expressed in several types of cancer. Thus, understanding the underlying mechanisms of regulation of PKM2 is of great value for targeted therapy for lung cancer.Patients and methodsSeventy-three lung adenocarcinoma patients were analyzed in our study. The expression levels of PKM2 were analyzed by immunohistochemistry on tissues. The effect of small ubiquitin-like modifier 1 (SUMO1) on PKM2 expression was investigated using Western blot assay and quantitative polymerase chain reaction. PKM2 SUMO1 modification was determined by in vitro and in vivo SUMOylation assays. 18F-deoxyglucose uptake and lactate production measurements were conducted to research the levels of glycolysis. The level of oxidative phosphorylation in cells was determined by cellular oxygen consumption rate measurements. Cell proliferation assays were carried out to confirm the growth ability of tumor cells.ResultsPKM2 was overexpressed in lung adenocarcinoma patients based on immunohistochemical staining. Patients with high PKM2 expression had reduced overall survival rate (P=0.017) and disease-free survival rate (P=0.027) compared with those with low PKM2 expression. SUMO1 promoted PKM2-dependent glycolysis. Western blotting analysis showed that SUMO1 knockdown in A549 cells led to a significant decrease in PKM2 protein expression. PKM2 could be covalently modified by SUMO1 at K336 (Lys336) site. SUMO1 modification of PKM2 at Lys-336 site increased glycolysis and promoted its cofactor functions. Moreover, PKM2 SUMO1 modification promoted the proliferation of A549 cells in vitro.ConclusionThis information is important in elucidating a new mechanism of regulation of PKM2, and suggested that SUMO1 modification of PKM2 could be a potential therapeutic target in lung cancer.

SUMO1 modification stabilizes CDK6 protein and drives the cell cycle and glioblastoma progression.

Ubiquitination governs oscillation of cyclin-dependent kinase (CDK) activity through a periodic degradation of cyclins for orderly cell cycle progression; however, the mechanism that maintains the constant CDK protein levels throughout the cell cycle remains unclear. Here we show that CDK6 is modified by small ubiquitin-like modifier-1 (SUMO1) in glioblastoma, and that CDK6 sumoylation stabilizes the protein and drives the cell cycle for the cancer development and progression. CDK6 is also a substrate of ubiquitin; however, CDK6 sumoylation at Lys 216 blocks its ubiquitination at Lys 147 and inhibits the ubiquitin-mediated CDK6 degradation. Throughout the cell cycle, CDK1 phosphorylates the SUMO-specific enzyme, ubiquitin-conjugating enzyme9 (UBC9) that in turn mediates CDK6 sumoylation during mitosis; CDK6 remain sumoylated in G1 phase and drives the cell cycle through G1/S transition. Thus, SUMO1-CDK6 conjugation constitutes a mechanism of cell cycle control and inhibition of this sumoylation pathway may provide a strategy for treatment of glioblastoma.

Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA)

Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugation to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis.

Lamin A tail modification by SUMO1 is disrupted by familial partial lipodystrophy–causing mutations

Lamin filaments are major components of the nucleoskeleton that bind LINC complexes and many nuclear membrane proteins. The tail domain of lamin A directly binds 21 known partners, including actin, emerin, and SREBP1, but how these interactions are regulated is unknown. We report small ubiquitin-like modifier 1 (SUMO1) as a major new posttranslational modification of the lamin A tail. Two SUMO1 modification sites were identified based on in vitro SUMOylation assays and studies of Cos-7 cells. One site (K420) matches the SUMO1 target consensus; the other (K486) does not. On the basis of the position of K486 on the lamin A Ig-fold, we hypothesize the SUMO1 E2 enzyme recognizes a folded structure-dependent motif that includes residues genetically linked to familial partial lipodystrophy (FPLD). Supporting this model, SUMO1-modification of the lamin A tail is reduced by two FPLD-causing mutations, G465D and K486N, and by single mutations in acidic residues E460 and D461. These results suggest a novel mode of functional control over lamin A in cells.

MDA5 is SUMOylated by PIAS2β in the upregulation of Type I interferon signaling

Retinoic acid-inducible protein I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are cytosolic viral RNA sensors that induce type I interferon production (IFN). In this study, we found that MDA5 undergoes inducible SUMOylation by small ubiquitin-like modifier-1 (SUMO-1) in response to polyI:C stimulation. Enhanced SUMOylation of MDA5 by exogenously expressed SUMO-conjugating enzyme Ubc9 correlated with upregulation of IFN expression and repressed virus replication. Conversely, overexpression of a SUMOylation-deficient mutant of Ubc9 or knockdown of endogenous Ubc9 reduced IFN production. Furthermore, we showed that PIAS2β, a SUMOylation E3 ligase, could specifically interact with and enhance the SUMOylation of MDA5. Consequently, PIAS2β knockdown reduced the SUMOylation of MDA5 and the IFN production. Collectively, these findings suggest that SUMO-1 modification of MDA5 possibly via PIAS2β may play a role in the MDA5-mediated IFN response to viral infections.

Adenosine Signaling Mediates SUMO-1 Modification of IκBα during Hypoxia and Reoxygenation

Small ubiquitin-like modifier 1 (SUMO-1) modification of IkappaBalpha has been described to actively participate in NFkappaB regulation. Following proteosomal degradation of IkappaBalpha, an auto-regulatory loop consisting of transcriptional activation of IkappaBalpha gene and SUMO-1 modification of newly synthesized IkappaBalpha proceeds. The SUMOylated IkappaBalpha form is resistant to signal-induced degradation, consequently halting NFkappaB activation. We describe a mechanistic model by which adenosine (Ado) signaling results in significant accumulation of SUMO-1 modified IkappaBalpha with subsequent attenuation of NFkappaB activation. Using models of hypoxia followed by reoxygenation (H/R), we have documented an H/R cycle-dependent increase in extracellular Ado correlating with increases in the cytoplasmic pool of IkappaBalpha/SUMO-1. We demonstrate a dose-dependent increase in IkappaBalpha/SUMO in cells treated with the general Ado receptor agonist NECA and abolished by Ado receptor antagonists. Experiments in cells exposed to cycles of H/R followed by hypoxia demonstrated differential patterns of SUMOylation and phosphorylation of IkappaBalpha, greatly impacting its proteosomal degradation by the 26 S proteasome. Assays targeting knockdown and overexpression of SUMO-1 demonstrated significant regulation of NFkappaB activation and NFkappaB-mediated gene transcription (interleukin-6). These results were confirmed in vivo using wild type and cd73 null mouse lung tissue. In summary, we present an endogenous mechanism by which cells and tissues acquire anti-inflammatory properties by recruiting a nondegradable form of IkappaBalpha, a major control point for NFkappaB activation via Ado signaling.

The mobility of Bach2 nuclear foci is regulated by SUMO-1 modification

The small ubiquitin-like modifier-1 (SUMO-1) modulates the functions of nuclear proteins by changing their structure and/or subnuclear localization. Several nuclear proteins form dynamic higher order nuclear structures, termed non-chromatin nuclear domains, which are involved in the regulation of nuclear function. However, the role that SUMO modification of the component proteins plays in the regulation of the activity and function of nuclear domains is unclear. Here we demonstrate that nuclear domains formed by Bach2, a transcription repressor, show restricted movement and undergo fusion events upon oxidative stress. Mutation of the SUMO-acceptor lysines in Bach2 alters the behavior of these nuclear foci and results in a decreased frequency of fusion events. We propose that SUMO modification is an important regulatory system for the mobility of the nuclear domains formed by Bach2.

A Method of Mapping Protein Sumoylation Sites by Mass Spectrometry Using a Modified Small Ubiquitin-like Modifier 1 (SUMO-1) and a Computational Program

Post-translational modification by small ubiquitin-like modifier 1 (SUMO-1) is a highly conserved process from yeast to humans and plays important regulatory roles in many cellular processes. Sumoylation occurs at certain internal lysine residues of target proteins via an isopeptide bond linkage. Unlike ubiquitin whose carboxyl-terminal sequence is RGG, the tripeptide at the carboxyl terminus of SUMO is TGG. The presence of the arginine residue at the carboxyl terminus of ubiquitin allows tryptic digestion of ubiquitin conjugates to yield a signature peptide containing a diglycine remnant attached to the target lysine residue and rapid identification of the ubiquitination site by mass spectrometry. The absence of lysine or arginine residues in the carboxyl terminus of mammalian SUMO makes it difficult to apply this approach to mapping sumoylation sites. We performed Arg scanning mutagenesis by systematically substituting amino acid residues surrounding the diglycine motif and found that a SUMO variant terminated with RGG can be conjugated efficiently to its target protein under normal sumoylation conditions. We developed a Programmed Data Acquisition (PDA) mass spectrometric approach to map target sumoylation sites using this SUMO variant. A web-based computational program designed for efficient identification of the modified peptides is described.

A Kruppel zinc finger of ZNF 146 interacts with the SUMO-1 conjugating enzyme UBC9 and is sumoylated in vivo

The OZF (ZNF146) protein is a 33 kDa Kruppel protein, composed solely of 10 zinc finger motifs. It is overexpressed in the majority of pancreatic cancers and in more than 80% of colorectal cancers. We have identified OZF interacting factors with a yeast two-hybrid screen. Half of the positive clones characterized encoded UBC9, the E2 enzyme involved in the covalent conjugation of the small ubiquitin-like modifier 1 (SUMO-1). SUMO-1 is a 17 kDa migrating protein that is conjugated to several proteins and has been reported to exhibit multiple effects, including modulation of protein stability, subcellular localization, and gene expression. In HeLa cells transfected with OZF and SUMO-1 expression vectors, immunoblot revealed a major band migrating at 50 kDa and a minor band at 67 kDa, corresponding to the attachment to OZF of one and two SUMO-1 proteins, respectively. The relative amount of the sumoylated proteins increased following transfection with a UBC9 expression vector. The presence of the sumoylated form in HeLa cells solely transfected by OZF indicates the physiological activity of the endogenous SUMO-1 conjugation pathway. Using deletion mutants, we showed that two SUMO-1 modification sites are located on the sixth zinc finger. Mutation of two lysine residues greatly reduced the amount of the sumoylated form of OZF though their surrounding sequences differ from the consensus sequence reported for most proteins modified by SUMO-1 conjugation. Despite the presence of the sixth zinc finger, an OZF mutant containing zinc fingers 1-6 was not modified by SUMO-1 and failed to interact with UBC9. Addition of zinc finger 7 restored SUMO-1 modification and UBC9 interaction and provides evidence that a region downstream of the target lysines is required for interaction with UBC9, in order to achieve SUMO-1 modification. This is the first report of in vivo conjugation of a SUMO-1 protein to a Kruppel zinc finger motif.

The Small Ubiquitin-like Modifier-1 (SUMO-1) Consensus Sequence Mediates Ubc9 Binding and Is Essential for SUMO-1 Modification

SUMO-1 is an ubiquitin-related protein that is covalently conjugated to a diverse assortment of proteins. The consequences of SUMO-1 modification include the regulation of protein-protein interactions, protein-DNA interactions, and protein subcellular localization. At present, very little is understood about the specific mechanisms that govern the recognition of proteins as substrates for SUMO-1 modification. However, many of the proteins that are modified by SUMO-1 interact directly with the SUMO-1 conjugating enzyme, Ubc9. These interactions suggest that Ubc9 binding may play an important role in substrate recognition as well as in substrate modification. The SUMO-1 consensus sequence (SUMO-1-CS) is a motif of conserved residues surrounding the modified lysine residue of most SUMO-1 substrates. This motif conforms to the sequence "PsiKXE," where Psi is a large hydrophobic residue, K is the lysine to which SUMO-1 is conjugated, X is any amino acid, and E is glutamic acid. In this study, we demonstrate that the SUMO-1-CS is a major determinant of Ubc9 binding and SUMO-1 modification. Mutating residues in the SUMO-1-CS abolishes both Ubc9 binding and substrate modification. These findings have important implications for how SUMO-1 substrates are recognized and for how SUMO-1 is ultimately transferred to specific lysine residues on these substrates.

Covalent Modification of p73α by SUMO-1: TWO-HYBRID SCREENING WITH p73 IDENTIFIES NOVEL SUMO-1-INTERACTING PROTEINS AND A SUMO-1 INTERACTION MOTIF

Two-hybrid screening in yeast with p73alpha isolated SUMO-1 (small ubiquitin-like modifier 1), the enzyme responsible for its conjugation, Ubc-9, and a number of novel SUMO-1-interacting proteins, including thymine DNA glycosylase, PM-Scl75, PIASx, PKY, and CHD3/ZFH. A subset of these proteins contain a common motif, hhXSXS/Taaa, where h is a hydrophobic amino acid and a is an acidic amino acid, that is shown to interact with SUMO-1 in the two-hybrid system. We show here that p73alpha, but not p73beta, can be covalently modified by SUMO-1. The major SUMO-1-modified residue in p73alpha is the C-terminal lysine (Lys(627)). The sequence surrounding this lysine conforms to a consensus SUMO-1 modification site b(X)XXhKXE, where b is a basic amino acid. SUMO-1-modified p73 is more rapidly degraded by the proteasome than unmodified p73, although SUMO-1 modification is not required for p73 degradation. SUMO-1 modification does not affect the transcriptional activity of p73alpha on an RGC-luciferase reporter gene in SK-N-AS cells. Instead, SUMO-1 modification may alter the subcellular localization of p73, because SUMO-1-modified p73 is preferentially found in detergent-insoluble fractions. Alternatively, it may modulate the interaction of p73 with other proteins that are substrates for SUMO-1 modification or which interact with SUMO-1, such as those identified here.