Phytosulfokine α (PSKα) delays senescence and reinforces SUMO1/SUMO E3 ligase SIZ1 signaling pathway in cut rose flowers (Rosa hybrida cv. Angelina).

Roses are widely used as cut flowers worldwide. Petal senescence confines the decorative quality of cut rose flowers, an impressively considerable economic loss. Herein, we investigated the SUMO1/SUMO E3 ligase SIZ1 signaling pathway during bud opening, and petal senescence of cut rose flowers. Our results exhibited that the higher expression of SUMO1 and SUMO E3 ligase SIZ1 during bud opening was accompanied by lower endogenous H2O2 accumulation arising from higher expression and activities of SOD, CAT, APX, and GR, promoting proline accumulation by increasing P5CS expression and activity and enhancing GABA accumulation by increasing GAD expression and activity. In harvested flowers, lower expressions of SUMO1 and SUMO E3 ligase SIZ1 during petal senescence were associated with higher endogenous H2O2 accumulation due to lower expression and activities of SOD, CAT, APX, and GR. Therefore, promoting the activity of the GABA shunt pathway as realized by higher expression and activities of GABA-T and SSADH accompanied by increasing OAT expression and activity for sufficiently supply proline in rose flowers during petal senescence might serve as an endogenous antisenescence mechanism for slowing down petals senescence by avoiding endogenous H2O2 accumulation. Following phytosulfokine α (PSKα) application, postponing petal senescence in cut rose flowers could be ascribed to higher expression of SUMO1 and SUMO E3 ligase SIZ1 accompanied by higher expression and activities of SOD, CAT, APX, and GR, higher activity of GABA shunt pathway as realized by higher expression and activities of GAD, GABA-T, and SSADH, higher expression and activities of P5CS and OAT for supplying proline and higher expression of HSP70 and HSP90. Therefore, our results highlight the potential of the PSKα as a promising antisenescence signaling peptide in the floriculture industry for postponing senescence and extending the vase life of cut rose flowers.

Rg3 promotes the SUMOylation of SERCA2a and corrects cardiac dysfunction in heart failure.

SUMOylation of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) has been shown to play a critical role in the abnormal Ca2+ cycle of heart failure. Ginsenoside Rg3 (Rg3), the main active constituent of Panax ginseng, exerts a wide range of pharmacological effects in cardiovascular diseases. However, the effect of Rg3 on abnormal Ca2+ homeostasis in heart failure has not been reported. In this study, we showed a novel role of Rg3 in the abnormal Ca2+ cycle in cardiomyocytes of mice with heart failure. Among mice undergoing transverse aortic constriction, animals that received Rg3 showed improvements in cardiac function and Ca2+ homeostasis, accompanied by increases in the SUMOylation level and SERCA2a activity. In an isoproterenol (ISO)-induced cell hypertrophy model, Rg3 reduced the ISO-induced Ca2+ overload in HL-1 cells. Gene knockout of SUMO1 in mice inhibited the cardioprotective effect of Rg3, and SUMO1 knockout mice that received Rg3 did not exhibit improved Ca2+ homeostasis in cardiomyocytes. Additionally, mutation of the SUMOylation sites of SERCA2a blocked the positive effect of Rg3 on the ISO-induced abnormal Ca2+ cycle in HL-1 cells, and was accompanied by an abnormal endoplasmic reticulum stress response and generation of ROS. Our data demonstrated that Rg3 has a positive effect on the abnormal Ca2+ cycle in the cardiomyocytes of mice with heart failure. SUMO1 is an important factor that mediates the protective effect of Rg3. Our findings suggest that drug intervention by regulating the SUMOylation of SERCA2a can provide a novel therapeutic strategy for the treatment of heart failure.

Time-Resolved Fluorescence Assays.

Fluorescence-based detection techniques are popular in high throughput screening due to sensitivity and cost-effectiveness. Four commonly used techniques exist, each with distinct characteristics. Fluorescence intensity assays are the simplest to run, but suffer the most from signal interference. Fluorescence polarization assays show less interference from the compounds or the instrument, but require a design that results in change of fluorophore-containing moiety size and usually have narrow assay signal window. Fluorescence resonance energy transfer (FRET) is commonly used for detecting protein-protein interactions and is constrained not by the sizes of binding partners, but rather by the distance between fluorophores. Time-resolved fluorescence resonance energy transfer (TR-FRET), an advanced modification of FRET approach utilizes special fluorophores with long-lived fluorescence and earns its place near the top of fluorescent techniques list by its performance and robustness, characterized by larger assay window and minimized compound spectral interference. TR-FRET technology can be applied in biochemical or cell-based in vitro assays with ease. It is commonly used to detect modulation of protein-protein interactions and in detection of products of biochemical reactions and cellular activities.

Motor neuron impairment mediated by a sumoylated fragment of the glial glutamate transporter EAAT2.

Dysregulation of glutamate handling ensuing downregulation of expression and activity levels of the astroglial glutamate transporter EAAT2 is implicated in excitotoxic degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). We previously reported that EAAT2 (a.k.a. GLT-1) is cleaved by caspase-3 at its cytosolic carboxy-terminus domain. This cleavage results in impaired glutamate transport activity and generates a proteolytic fragment (CTE) that we found to be post-translationally conjugated by SUMO1. We show here that this sumoylated CTE fragment accumulates in the nucleus of spinal cord astrocytes of the SOD1-G93A mouse model of ALS at symptomatic stages of disease. Astrocytic expression of CTE, artificially tagged with SUMO1 (CTE-SUMO1) to mimic the native sumoylated fragment, recapitulates the nuclear accumulation pattern of the endogenous EAAT2-derived proteolytic fragment. Moreover, in a co-culture binary system, expression of CTE-SUMO1 in spinal cord astrocytes initiates extrinsic toxicity by inducing caspase-3 activation in motor neuron-derived NSC-34 cells or axonal growth impairment in primary motor neurons. Interestingly, prolonged nuclear accumulation of CTE-SUMO1 is intrinsically toxic to spinal cord astrocytes, although this gliotoxic effect of CTE-SUMO1 occurs later than the indirect, noncell autonomous toxic effect on motor neurons. As more evidence on the implication of SUMO substrates in neurodegenerative diseases emerges, our observations strongly suggest that the nuclear accumulation in spinal cord astrocytes of a sumoylated proteolytic fragment of the astroglial glutamate transporter EAAT2 could participate to the pathogenesis of ALS and suggest a novel, unconventional role for EAAT2 in motor neuron degeneration.

Membrane lipid modification by docosahexaenoic acid (DHA) promotes the formation of α-synuclein inclusion bodies immunopositive for SUMO-1 in oligodendroglial cells after oxidative stress.

α-Synuclein (α-syn) is the major constituent of Lewy bodies and glial cytoplasmic inclusions which are pathological hallmarks of neurodegenerative disorders like Parkinson's disease or multiple system atrophy (MSA), respectively. It accumulates and aggregates during the pathogenic process, and missense mutations, such as A53T, are increasing its probability of aggregate formation. Furthermore, α-syn interacts with polyunsaturated fatty acids, and this interaction may promote the oligomerization process. To investigate whether membrane lipid modification by docosahexaenoic acid (DHA) modifies the aggregation process of α-syn in oligodendroglial cells, we have used OLN-93 cells stably expressing the human α-syn A53T mutation. Cells were supplemented with DHA (25 µM) for 3 days and then subjected to oxidative stress (OS) exerted by hydrogen peroxide. The data show that modification of the oligodendroglial cell membranes by DHA followed by OS caused the formation of fibrillary α-syn inclusions, a decrease in α-syn solubility, and an increase in phosphorylation at serine 129, which has been suggested to play a proaggregatory role. The aggregates contain αB-crystallin and ubiquitinated proteins and SUMO-1 immunoreactivity. SUMO-1 has been implicated in protein aggregation and identified as a constituent in inclusion bodies in MSA. Hence, membrane lipid modification in oligodendroglial cells promotes the formation of α-syn inclusion bodies resembling protein aggregates in neurodegenerative disease. This effect is not only attributable to the A53T mutation but also is observable in OLN cells expressing wild-type α-syn.

A simple in situ cell-based sumoylation assay with potential application to drug screening.

We found that the small ubiquitin-related modifier (SUMO) conjugation reaction can be visualized simply by incubating green fluorescent protein (GFP)-SUMO-1 with permeabilized cells in the presence of ATP for 15 min. Neither special equipment for protein purification nor highly developed skills for recombinant technologies is required, making the assay potentially applicable to large-scale drug-screening strategies for the identification of drugs that can inhibit or enhance SUMOylation.

SUMO fusion facilitates expression and purification of garlic leaf lectin but modifies some of its properties.

Over expression of lectin genes in E. coli often gives inclusion bodies that are solubilised to characterize lectins. We made N-terminal fusion of the Allium sativum leaf agglutinin (ASAL) with SUMO (small ubiquitin related modifier) peptide. The SUMO peptide allowed expression of the recombinant lectin in E. coli, predominantly in soluble form. The soluble fusion protein could be purified by immobilized metal affinity column (IMAC), followed by size exclusion chromatography. The SUMO protease failed to cleave the SUMO peptide from ASAL. This may be due to steric hindrance caused by the homodimer structure of the chimeric ASAL. Some properties like dimerization, haemagglutination and insecticidal properties of the recombinant SUMO-ASAL fusion protein were comparable to the plant derived native lectin. However, glycan array analysis revealed that the carbohydrate binding specificity of the recombinant SUMO-ASAL was altered. Further, the fusion protein was not toxic to E. coli (native ASAL exhibited toxicity). The recombinant lectin was more thermo-labile as compared to the native lectin. Three important findings of this study are: (1) sugar specificity of ASAL can be altered by amino-terminal fusion; (2) anti-E. coli activity of ASAL can be eliminated by N-terminal SUMO fusion and (3) SUMO-ASAL may be a preferred candidate insecticidal protein for the development of transgenic plants.

p300 provides a corepressor function by cooperating with YY1 and HDAC3 to repress c-Myc.

We showed earlier that p300/CBP plays an important role in G1 progression by negatively regulating c-Myc and thereby preventing premature G1 exit. Here, we have studied the mechanism by which p300 represses c-Myc and show that in quiescent cells p300 cooperates with histone deacetylase 3 (HDAC3) to repress transcription. p300 and HDAC3 are recruited to the upstream YY1-binding site of the c-Myc promoter resulting in chromatin deacetylation and repression of c-Myc transcription. Consistent with this, ablation of p300, YY1 or HDAC3 expression results in chromatin acetylation and induction of c-Myc. These three proteins exist as a complex in vivo and form a multiprotein complex with the YY1-binding site in vitro. The C-terminal region of p300 is both necessary and sufficient for the repression of c-Myc. These and other results suggest that in quiescent cells the C-terminal region of p300 provides corepressor function and facilitates the recruitment of p300 and HDAC3 to the YY1-binding site and represses the c-Myc promoter. This corepressor function of p300 prevents the inappropriate induction of c-Myc and S phase.

PIASy-mediated sumoylation of Yin Yang 1 depends on their interaction but not the RING finger.

As a multifunctional protein, Yin Yang 1 (YY1) has been demonstrated to regulate both gene expression and protein posttranslational modifications. However, gaps still exist in our knowledge of how YY1 can be modified and what the consequences of its modifications are. Here we report that YY1 protein can be sumoylated both in vivo and in vitro. We have identified lysine 288 as the major sumoylation site of YY1. We also discovered that PIASy, a SUMO E3 ligase, is a novel YY1-interacting protein and can stimulate the sumoylation of YY1 both in vitro and in vivo. Importantly, the effects of PIASy mutants on in vivo YY1 sumoylation correlate with the YY1-PIASy interaction but do not depend on the RING finger domain of PIASy. This regulation is unique to YY1 sumoylation because PIASy-mediated p53 sumoylation still relies on the integrity of PIASy, which is also true of all of the previously identified substrates of PIASy. In addition, PIASy colocalizes with YY1 in the nucleus, stabilizes YY1 in vivo, and differentially regulates YY1 transcriptional activity on different target promoters. This study demonstrates that YY1 is a target of SUMOs and reveals a novel feature of a SUMO E3 ligase in the PIAS family that selectively stimulates protein sumoylation independent of the RING finger domain.

Modulation of HIV-1 Rev protein abundance and activity by polyubiquitination with unconventional Lys-33 branching.

The HIV-1 Rev protein plays a key role in virus replication by allowing export to the cytoplasm of unspliced or singly-spliced RNAs. In this report, we investigated whether Rev is modified by ubiquitination or sumoylation. Whereas no evidence of sumoylation was obtained, transient expression experiments showed that ubiquitin conjugates to Rev as high molecular weight polyubiquitin chains. Mutation of the three lysine residues of Rev showed that the site of ubiquitin conjugation is Lys-115. Experiments with ubiquitin mutants including a single lysine at every seven possible position indicated that branching of the polyubiquitin chains mainly involves Lys-33. Mutation of Rev Lys-115 to arginine reduces markedly the steady state amount of the protein, but does not impair its ability to export RNA via the Rev response element. These observations support the notion that polyubiquitination of Rev stabilizes the viral protein but hinders its activity.

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.

SUMO modification of the Ets-related transcription factor ERM inhibits its transcriptional activity.

A variety of transcription factors are post-translationally modified by SUMO, a 97-residue ubiquitin-like protein bound covalently to the targeted lysine. Here we describe SUMO modification of the Ets family member ERM at positions 89, 263, 293, and 350. To investigate how SUMO modification affects the function of ERM, Ets-responsive intercellular adhesion molecule 1 (ICAM-1) and E74 reporter plasmids were employed to demonstrate that SUMO modification causes inhibition of ERM-dependent transcription without affecting the subcellular localization, stability, or DNA-binding capacity of the protein. When the adenoviral protein Gam1 or the SUMO protease SENP1 was used to inhibit the SUMO modification pathway, ERM-dependent transcription was de-repressed. These results demonstrate that ERM is subject to SUMO modification and that this post-translational modification causes inhibition of transcription-enhancing activity.

Caspase-8 sumoylation is associated with nuclear localization.

The cysteine protease caspase-8 plays a pivotal role in the initiation of different apoptotic pathways and controls the maturation and differentiation of various cell types including neurons, fibroblasts and lymphocytes. Specific substrates of caspase-8 are present in both the cytoplasm and the nucleus, which may determine the ultimate biological effect of caspase-8. However, the mechanisms regulating the cellular localization of caspase-8 are still unknown. We show here that, in contrast to other caspases such as caspase-9 and -3, caspase-8 can be sumoylated at lysine 156. This sumoylation (i) is associated with the nuclear localization of caspase-8 and (ii) did not impair caspase-8 activation.

Serum response factor is modulated by the SUMO-1 conjugation system.

Serum stimulation leads to activation of the serum response factor (SRF)-mediated transcription of immediate-early genes such as c-fos via various signal transduction pathways. We have previously reported that promyelocytic leukemia protein (PML) is involved in the transcriptional regulation by SRF. PML is one of the well-known substrates for modification by small ubiquitin-related modifier-1 (SUMO-1) and several SUMO-1-modified proteins associate with PML. Here, we report that SRF is modified by SUMO-1 chiefly at lysine(147) within the DNA-binding domain. Substitution of this target lysine for alanine did not affect the translocation of SRF to PML-nuclear bodies. The SRF mutant augmented the transcriptional activity under Rho A-stimulated condition but not under serum-starved condition, suggesting that activated SRF is suppressed by its sumoylation. These data support the transcriptional role of SUMO-1 conjugating system in cellular serum response.

Sumoylation of Smad4, the common Smad mediator of transforming growth factor-beta family signaling.

Transforming growth factor-beta (TGF-beta) and TGF-beta-related factors regulate cell growth, differentiation, and apoptosis, and play key roles in normal development and tumorigenesis. TGF-beta family-induced changes in gene expression are mediated by serine/threonine kinase receptors at the cell surface and Smads as intracellular effectors. Receptor-activated Smads combine with a common Smad4 to translocate into the nucleus where they cooperate with other transcription factors to activate or repress transcription. The activities of the receptor-activated Smads are controlled by post-translational modifications such as phosphorylation and ubiquitylation. Here we show that Smad4 is modified by sumoylation. Sumoylation of Smad4 was enhanced by the conjugating enzyme Ubc9 and members of the PIAS family of SUMO ligases. A major sumoylation site in Smad4 was localized to Lys-159 in its linker segment with an additional site at Lys-113 in the MH-1 domain. Increased sumoylation in the presence of the PIASy E3 ligase correlated with targeting of Smad4 to subnuclear speckles that contain SUMO-1 and PIASy. Replacement of lysines 159 and 113 by arginines or increased sumoylation enhanced the stability of Smad4, and transcription in mammalian cells and Xenopus embryos. These observations suggest a role for Smad4 sumoylation in the regulation of TGF-beta signaling through Smads.

Role of two residues proximal to the active site of Ubc9 in substrate recognition by the Ubc9.SUMO-1 thiolester complex.

The small ubiquitin-like modifier SUMO-1 is covalently attached to lysine residues on target proteins by a specific conjugation pathway involving the E1 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-1 thiolester complex catalyzes the formation of an isopeptide bond between SUMO-1 and the epsilon-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 (Ubls), 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(100) and Lys(101) 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.