A SUMO1-Derived Peptide Targeting SUMO-Interacting Motif Inhibits α-Synuclein Aggregation.

The accumulation of α-synuclein amyloid fibrils in the brain is linked to Parkinson's disease and other synucleinopathies. The intermediate species in the early aggregation phase of α-synuclein are involved in the emergence of amyloid toxicity and considered to be the most neurotoxic. The N-terminal region flanking the non-amyloid-ß component domain of α-synuclein has been implicated in modulating its aggregation. Herein, we report the development of a SUMO1-derived peptide inhibitor (SUMO1(15-55)), which targets two SUMO-interacting motifs (SIMs) within this aggregation-regulating region and suppresses α-synuclein aggregation. Molecular modeling, site-directed mutagenesis, and binding studies are used to elucidate the mode of interaction, namely, via the binding of either of the two SIM sequences on α-synuclein to a putative hydrophobic binding groove on SUMO1(15-55). Subsequent studies show that SUMO1(15-55) also reduces α-synuclein-induced cytotoxicity in cell-based and Drosophila disease models.

Phosphorylation and SUMOylation of CRMP2 regulate the formation and maturation of dendritic spines.

The posttranslational modifications of CRMP2 play an important role in axon outgrowth, cell polarization and dendritic morphogenesis. However, whether CRMP2 and its posttranslational modifications are involved in dendritic spine development specifically is not completely clear. Here, we show that CRMP2 can promote the formation and maturation of dendritic spines in cultured hippocampal neurons. Overexpression of CRMP2 results in an increase in the density of spines especially the mushroom-shape spines. The amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs) are both enhanced and the intensity of PSD95 is strengthened in the neurons with CRMP2 overexpression. Furthermore, dephosphorylation of CRMP2 at Thr514 and deSUMOylation at Lys374 can further promote the formation and maturation of dendritic spines, whereas, no cross-talk is found between these two posttranslational modifications in the regulation of dendritic spine formation and maturation. Taken together, our data support a model in which phosphorylation and SUMOylation modification of CRMP2 independently promote the formation and maturation of dendritic spines and participate in the process of dendritic spine plasticity.

Recombinant Production and Characterization of SAC, the Core Domain of Par-4, by SUMO Fusion System.

Prostate apoptosis response-4 (Par-4), an anticancer protein that interacts with cell surface receptor GRP78, can selectively suppress proliferation and induce apoptosis of cancer cells. The core domain of Par-4 (aa 137-195), designated as SAC, is sufficient to inhibit tumor growth and metastasis without harming normal tissues and organs. Nevertheless, the anticancer effects of SAC have not been determined in ovarian cancer cells. Here, we developed a novel method for producing native SAC in Escherichia coli using a small ubiquitin-related modifier (SUMO) fusion system. This fusion system not only greatly improved the solubility of target protein but also enhanced the expression level of SUMO-SAC. After purified by Ni-NTA affinity chromatography, SUMO tag was cleaved from SUMO-SAC fusion protein using SUMO protease to obtain recombinant SAC. Furthermore, we simplified the purification process by combining the SUMO-SAC purification and SUMO tag cleavage into one step. Finally, the purity of recombinant SAC reached as high as 95% and the yield was 25 mg/L. Our results demonstrated that recombinant SAC strongly inhibited proliferation and induced apoptosis in ovarian cancer cells SKOV-3. Immunofluorescence analysis and competitive binding reaction showed that recombinant SAC could specifically induce apoptosis of SKOV-3 cells through combination with cell surface receptor, GRP78. Therefore, we have developed an effective strategy for expressing bioactive SAC in prokaryotic cells, which supports the application of SAC in ovarian cancer therapy.

SUMO-1 delays neuronal damage in the spinal cord following ischemia/reperfusion.

The present study investigated the protective effects of small ubiquitin-like modifier 1 (SUMO-1) on spinal cord ischemic damage in rabbits. A trans­activator of transcription (Tat)­SUMO­1 fusion protein was prepared, and transient spinal cord ischemia was induced by occlusion of the abdominal aorta for 15 min. Vehicle (glycerol) or 1 mg/kg Tat-1-SUMO­1 was administered intraperitoneally to the rabbits immediately following ischemia/reperfusion. Administration of Tat-SUMO-1 did not lead to significant alterations in arterial blood gases [partial pressure (Pa)CO2 and PaO2], pH, or blood glucose levels prior to ischemia, 10 min after occlusion or 10 min after reperfusion. Mean arterial pressure was significantly decreased only during occlusion. Motor behaviors were assessed at 24, 48 and 72 h after ischemia/reperfusion using Tarlov's criteria. Administration of Tat­SUMO­1 significantly improved Tarlov scores 24 h after ischemia/reperfusion and the number of cresyl violet positive neurons was significantly increased in the ventral horn of the spinal cord compared with the vehicle­treated group. However, Tarlov scores were consistently decreased at 48 and 72 h after ischemia/reperfusion in the Tat­SUMO­1­treated group, and Tarlov scores and the number of cresyl violet positive neurons were not significantly different between the vehicle­ and Tat­SUMO­1­treated groups after 72 h. Tat-SUMO­1 administration significantly ameliorated a reduction in Cu, Zn­superoxide dismutase activity and an increase in lipid peroxidation 24 h after ischemia/reperfusion; however, these effects were not present at 72 h. These results suggested that Tat­SUMO­1 may delay, although not protect against, neuronal death by regulating oxidative stress in the ventral horn of the spinal cord and that combination therapy using Tat­SUMO­1 with other compounds may provide a therapeutic approach to decrease neuronal damage.

The Bcl-2/Bcl-xL inhibitor BH3I-2' affects the dynamics and subcellular localization of sumoylated proteins.

Sumoylation modulates many proteins implicated in apoptosis such as Fas, TNFR1, Daxx, p53 and its regulator MDM2. Some of these proteins, such as DRP-1, are involved in the intrinsic apoptosis pathway. The intrinsic pathway is regulated at the mitochondrial level by the Bcl-2 family of proteins. The small-molecule inhibitor BH3I-2' binds to the hydrophobic groove of the BH3 domain of anti-apoptotic proteins Bcl-xL and Bcl-2. Following treatment with this inhibitor in various experimental conditions, we observed decreased levels of detergent-soluble SUMO-1, an increase in the relative levels of detergent-insoluble sumoylated proteins, or both. Accordingly, immunofluorescence microscopy revealed that the relative numbers and intensities of endogenously or exogenously expressed SUMO-1 foci in the nucleus were increased following BH3I-2' treatment. MG132 caused a large increase in steady-state levels of SUMO-1 and of sumoylated proteins, and this was especially true for detergent-insoluble proteins. The conjugation-incompetent GG-to-AA SUMO-1 mutant, which did not form nuclear foci, was only present in the detergent-soluble lysate fraction and was insensitive to BH3I-2', implying that BH3I-2' specifically affects SUMO in its conjugated form. Finally, BH3I-2' had similar effects on SUMO-2 and SUMO-3 as it had on SUMO-1. In conclusion, BH3I-2' causes an intracellular redistribution of sumoylated proteins, more specifically their targeting to PML and non-PML nuclear bodies in which they may be degraded by the proteasome. Interestingly, knocking down Bcl-2 also altered levels of sumoylated proteins and their presence in detergent-insoluble compartments, confirming the role of Bcl-2 as a modulator of the sumoylation pathway.

Identification of Chinese cabbage sentrin as a suppressor of Bax-induced cell death in yeast.

Studies into the cell death program termed apoptosis have resulted in new information regarding how cells control and execute their own demise, including insights into the mechanism by which death-preventing factors can inhibit Bax-induced caspase activation. We investigated high temperature stress-induced cell death in Brassica rapa. Using a yeast functional screening from a Brassica rapa cDNA library, the BH5-127 EST clone encoding an apoptotic suppressor peptide was identified. However, a phylogenic tree showed that BH5-127 clusters within a clade containing SUMO-1 (Small Ubiquitin-like Modifier- 1). BH5-127 was confirmed similar to have function to SUMO-1 as Fas suppression. Expression of BH5-127 showed that substantial suppression of cell death survived on SD-galactose-Leu--Ura- medium. The results suggest that BrSE (Brassica rapa Sentrin EST, BH5-127) is one of the important regulatory proteins in programming cell death, especially in the seedling stage of Chinese cabbage.

Host cell sumoylation level influences papillomavirus E2 protein stability.

The stability of papillomavirus E2 proteins is regulated by proteasomal degradation, and regulation of degradation could contribute to the higher expression levels of E2 proteins observed in suprabasal layers of differentiated skin. We have recently shown that the E2 proteins are modified by sumoylation [Wu Y-C, Roark AA, Bian X-L, Wilson, VG (2008) Virol 378:329-338], and that sumoylation levels are up-regulated during keratinocyte differentiation [Deyrieux AF, Rosas-Acosta G, Ozbun MA, Wilson VG (2007) J Cell Sci 120:125-136]. These observations, coupled with the known ability of sumoylation to prevent proteasomal degradation of certain proteins, suggested that this modification might contribute to stabilizing E2 proteins in suprabasal keratinocytes. Conditions that increased overall sumoylation were found to increase the intracellular amounts of the HPV11, 16, and 18 E2 proteins. No effect of sumoylation was seen on E2 transcripts, and the increased levels of E2 proteins resulted from a greatly increased half-life for the E2 proteins. In vitro studies confirmed that sumoylation could block the proteasomal degradation of the 16E2 protein. Interestingly, this stabilization effect was indirect as it did not require sumoylation of 16E2 itself and must be acting through sumoylation of a cellular target(s). This sumoylation-dependent, indirect stabilization of E2 proteins is a novel process that may couple E2 levels to changes in the cellular environment. Specifically, our results suggest that the levels of papillomavirus E2 protein could be up-regulated in differentiating keratinocytes in response to the increased overall sumoylation that accompanies differentiation.

Nuclear localisation of endogenous SUMO-1-modified PDGF-C in human thyroid tissue and cell lines.

We investigated post-translational modification and subcellular localisation of endogenous platelet-derived growth factor-C (PDGF-C) in human thyroid papillary carcinomas (PTC), non-neoplastic thyroid tissues, and a selection of cultured cell lines. PDGF-C expressed nuclear localisation in 95% of all tested cell types in culture and in 10% of the thyrocytes from both PTC and non-neoplastic tissue. The cell lines expressed two forms of full-length PDGF-C, approximately 39 and approximately 55 kDa, in cell membrane and cytosol, while the approximately 55 kDa form dominated in the nucleus where it was partly chromatin-associated. The approximately 55 kDa form was post-translationally modified by SUMO-1. The putative PDGF-C SUMOylation site is the surface exposed (314)lysine part of a positively charged loop ((312)RPKTGVRGLHK(322)) with characteristics of a nuclear localisation signal. The tissue thyrocytes expressed a non-SUMOylated approximately 43 kDa and the 55 kDa PDGF-C. The SUMO-1 modified approximately 55 kDa PDGF-C expression was low in PTC where the approximately 43 kDa PDGF-C dominated. This is in contrast to non-neoplastic tissue and cultured cells where the SUMOylated approximately 55 kDa PDGF-C was strongly expressed. Our data provide novel evidence for nuclear localisation of PDGF-C, post-translational modification by SUMOylation and the expression of a novel form of PDGF-C in human papillary thyroid carcinomas.

SUMO-1 enhancing the p53-induced HepG2 cell apoptosis.

In order to investigate the effect of small ubiquitin-like modifier-1 (SUMO-1) on the p53-induced HepG2 cell apoptosis, HepG2 cells were transfected by recombinant plasmids as pwtp53, pMDM2 and pSUMO-1 respectively. Western blot was employed to detect the protein expression of the transfected recombinant plasmids and the rate of apoptosis was measured by flow cytometry. The results showed that in cells transfected with pwtp53 and pwtp53 + pSUMO-1, the apoptosis rate was (16.79+/-1.62) % and (18.15+/-1.36) % respectively, while transfected with pwtp53+ pMDM2, the rate was decreased to (5.17+/-1.23) %. The apoptosis rate was (14.06+/-1.84) % in the cells transfected with pwtp53+pMDM2+pSUMO-1, significantly higher than that in the cells Transfected with pwtp53+pMDM2 (P<0.01). The apoptosis rates in the cells were all less than 2 % and had no significant difference among the groups. It was suggested that in the HepG2 cells, SUMO-1 can increase the apoptosis induced by wild-type p53 through binding to p53 protein, posttranslational modification and inhibiting the p53 degradation by MDM2.

Inhibition of HIV-1 replication by cell-penetrating peptides binding Rev.

New therapeutic agents able to block HIV-1 replication are eagerly sought after to increase the possibilities of treatment of resistant viral strains. In this report, we describe a rational strategy to identify small peptide sequences owning the dual property of penetrating within lymphocytes and of binding to a protein target. Such sequences were identified for two important HIV-1 regulatory proteins, Tat and Rev. Their association to a stabilizing domain consisting of human small ubiquitin-related modifier-1 (SUMO-1) allowed the generation of small proteins named SUMO-1 heptapeptide protein transduction domain for binding Tat (SHPT) and SUMO-1 heptapeptide protein transduction domain for binding Rev (SHPR), which are stable and efficiently penetrate within primary lymphocytes. Analysis of the antiviral activity of these proteins showed that one SHPR is active in both primary lymphocytes and macrophages, whereas one SHPT is active only in the latter cells. These proteins may represent prototypes of new therapeutic agents targeting the crucial functions exerted by both viral regulatory factors.

Activation of transforming growth factor-beta signaling by SUMO-1 modification of tumor suppressor Smad4/DPC4.

Smads are important intracellular effectors in signaling pathways of the transforming growth factor-beta (TGF-beta) superfamily. Upon activation by TGF-beta, receptor-phosphorylated Smads form a complex with tumor suppressor Smad4/DPC4, and the Smad complexes then are imported into the nucleus. Although diverse pathways regulate the activity and expression of receptor-phosphorylated and inhibitory Smads, cellular factors modulating the activity of the common Smad4 remain unidentified. Here we describe the involvement of the small ubiquitin-like modifier-1 (SUMO-1) conjugation pathway in regulating the growth inhibitory and transcriptional responses of Smad4. The MH1 domain of Smad4 was shown to associate physically with Ubc9, the ubiquitin carrier protein (E2) conjugating enzyme in sumoylation. In cultured cells, Smad4 is modified by SUMO-1 at the endogenous level. The sumoylation sites were identified as two evolutionarily conserved lysine residues, Lys-113 and Lys-159, in the MH1 domain. We found that the mutations at Lys-113 and Lys-159 did not alter the ability of Smad4 to form a complex with Smad2 and FAST on the Mix.2 promoter. Importantly, SUMO-1 overexpression enhanced TGF-beta-induced transcriptional responses. These findings identify sumoylation as a unique mechanism to modulate Smad4-dependent cellular responses.