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.

SUMO-1 gene transfer improves cardiac function in a large-animal model of heart failure.

Recently, the impact of small ubiquitin-related modifier 1 (SUMO-1) on the regulation and preservation of sarcoplasmic reticulum calcium adenosine triphosphatase (SERCA2a) function was discovered. The amount of myocardial SUMO-1 is decreased in failing hearts, and its knockdown results in severe heart failure (HF) in mice. In a previous study, we showed that SUMO-1 gene transfer substantially improved cardiac function in a murine model of pressure overload-induced HF. Toward clinical translation, we evaluated in this study the effects of SUMO-1 gene transfer in a swine model of ischemic HF. One month after balloon occlusion of the proximal left anterior descending artery followed by reperfusion, the animals were randomized to receive either SUMO-1 at two doses, SERCA2a, or both by adeno-associated vector type 1 (AAV1) gene transfer via antegrade coronary infusion. Control animals received saline infusions. After gene delivery, there was a significant increase in the maximum rate of pressure rise [dP/dt(max)] that was most pronounced in the group that received both SUMO-1 and SERCA2a. The left ventricular ejection fraction (LVEF) improved after high-dose SUMO-1 with or without SERCA2a gene delivery, whereas there was a decline in LVEF in the animals receiving saline. Furthermore, the dilatation of LV volumes was prevented in the treatment groups. SUMO-1 gene transfer therefore improved cardiac function and stabilized LV volumes in a large-animal model of HF. These results support the critical role of SUMO-1 in SERCA2a function and underline the therapeutic potential of SUMO-1 for HF patients.

Expression, purification and preliminary NMR characterization of isotopically labeled wild-type human heterotrimeric G protein αi1.

Molecular-level investigation of proteins is increasingly important to researchers trying to understand the mechanisms of signal transmission. Heterotrimeric G proteins control the activation of many critical signal transmission cascades and are also implicated in numerous diseases. As part of a longer-term investigation of intramolecular motions in RGS and Gα proteins in their apo and complexed forms, we have successfully developed a protocol for preparing milligram quantities of highly purified, isotopically labeled wild-type human Gα(i1) (hGα(i1)) subunit for NMR studies. High levels of expression in Escherichia coli can be attributed to the use of the SUMO fusion protein system, a bacterial strain that produces rare codons, supplementation of minimal medium with small quantities of isotopically labeled rich medium and a lowered induction temperature. Purification of hGα(i1) utilized affinity and size exclusion chromatography, and protein activity was confirmed using fluorescence-based GTP-binding studies. Preliminary NMR analysis of hGα(i1) has shown that high-quality spectra can be obtained at near-physiological temperatures, whereas lower temperature spectra display numerous weak and broadened peaks, providing preliminary evidence for widespread µs-ms timescale exchange. In an effort to further optimize the NMR spectra we prepared a truncated form of hGα(i1) (hGα(i1)-Δ31) in which the 31-residue unstructured N-terminus was removed. This resulted in further improvements in spectral quality by eliminating high-intensity peaks that obscured resonances from structured segments of the protein. We plan to use hGα(i1)-Δ31 in future investigations of protein dynamics by NMR spectroscopy to gain insight into the role of these motions in RGS/Gα binding selectivity.

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.

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.

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.

Transcription factor sumoylation and factor YY1 serve to modulate mouse amelogenin gene expression.

Amelogenin proteins are essential in the control of enamel biomineralization and the amelogenin gene therefore is spatiotemporally regulated to ensure proper amelogenin protein expression. In this study, we examined the role of sumoylation to alter CCAAT/enhancer-binding protein alpha (C/EBPalpha) activity, and performed a search using a protein/DNA array system for other proteins that act co-operatively with C/EBPalpha to alter amelogenin expression. We observed that C/EBPalpha was modified by sumoylation, and that this modification played an indirect inhibitory role on the regulation of C/EBPalpha activity which appeared to act through other transcription factors. The protein/DNA array allowed us to single out the transcription factor, YY1, which acts in the absence of direct DNA binding to repress both the basal amelogenin promoter activity and C/EBPalpha-mediated transactivation. Taken together, these pathways may account for part of the physiological modulation of the amelogenin gene expression in accordance with tooth developmental and enamel biomineralization requirements.

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.