SUMO regulates the cytoplasmonuclear transport of its target protein Daxx.

It is known that Fas death domain-associated protein (Daxx) possesses both putative nuclear and cytoplasmic functions. However, the nuclear transport mechanism is largely unknown. This study examined the nuclear location signal (NLS) of Daxx and whether the nuclear transport of Daxx was mediated by small ubiquitin-related modifier (SUMO). Two NLS motifs of Daxx, leucine (L)-rich nuclear export signal (NES)-like motif (188IXXLXXLLXL197) and C-terminal lysine (K) rich NLS2 (amino acids 627-634) motif, were identified and the K630 and K631 on the NLS2 motif were characterized as the major sumoylation sites of Daxx by in vitro sumoylation analysis. Proteins of inactive SUMO (SUMO-delta), a sumoylation-incompetent mutant, and Daxx NLS mutants (Daxx-NES(mut) and Daxx NLS2(mut)) were dispersed in cytoplasm. The cytoplasmic dispersed Daxx mutants could be relocalized to nucleus by cotransfection with active SUMO, but not with inactive SUMO-delta, demonstrating the role of SUMO on regulating the cytoplasmonuclear transport of Daxx. However, inactive SUMO-delta could also be relocalized to nucleus during cotransfection with wild-type Daxx, suggesting that SUMO regulation of the cytoplasmonuclear transport of its target protein Daxx does not need covalent modification. This study shows that cytoplasmic SUMO has a biological role in enhancing the cytoplasmonuclear transport of its target protein Daxx and it may be done through the non-sumoylation interactions.

Crystal structures of the human SUMO-2 protein at 1.6 A and 1.2 A resolution: implication on the functional differences of SUMO proteins.

The SUMO proteins are a class of small ubiquitin-like modifiers. SUMO is attached to a specific lysine side chain on the target protein via an isopeptide bond with its C-terminal glycine. There are at least four SUMO proteins in humans, which are involved in protein trafficking and targeting. A truncated human SUMO-2 protein that contains residues 9-93 was expressed in Escherichia coli and crystallized in two different unit cells, with dimensions of a=b=75.25 A, c=29.17 A and a=b=74.96 A, c=33.23 A, both belonging to the rhombohedral space group R3. They diffracted X-rays to 1.6 A and 1.2 A resolution, respectively. The structures were determined by molecular replacement using the yeast SMT3 protein as a search model. Subsequent refinements yielded R/Rfree values of 0.169/0.190 and 0.119/0.185, at 1.6 A and 1.2 A, respectively. The peptide folding of SUMO-2 consists of a half-open beta-barrel and two flanking alpha-helices with secondary structural elements arranged as betabetaalphabetabetaalphabeta in the sequence, identical to those of ubiquitin, SMT3 and SUMO-1. Comparison of SUMO-2 with SUMO-1 showed a surface region near the C terminus with significantly different charge distributions. This may explain their distinct intracellular locations. In addition, crystal-packing analysis suggests a possible trimeric assembly of the SUMO-2 protein, of which the biological significance remains to be determined.

Post-translational modification of Rta of Epstein-Barr virus by SUMO-1.

Epstein-Barr virus (EBV) expresses an immediate-early protein, Rta, to activate the transcription of EBV lytic genes and the lytic cycle. This work identifies Ubc9 and PIAS1 as binding partners of Rta in a yeast two-hybrid screen. These bindings are verified by glutathione S-transferase pull-down assay, coimmunoprecipitation, and confocal microscopy. The interactions appear to cause Rta sumoylation, because not only can Rta be sumoylated in vitro but also sumoylated Rta can be detected in P3HR1 cells following lytic induction and in 293T cells after transfecting plasmids that express Rta and SUMO-1. Moreover, PIAS1 stimulates conjugation of SUMO-1 to Rta, thus acting as an E3 ligase. Furthermore, transfecting plasmids that express Ubc9, PIAS1, and SUMO-1 increases the capacity of Rta to transactivate the promoter that includes an Rta response element, indicating that the modification by SUMO-1 increases the transactivation activity of Rta. This study reveals that Rta is sumoylated at the Lys-19, Lys-213, and Lys-517 residues and that SUMO-1 conjugation at the Lys-19 residue is crucial for enhancing the transactivation activity of Rta. These results indicate that sumoylation of Rta may be important in EBV lytic activation.

The use of a distal occlusion balloon protection device in acute coronary syndrome.

We report our early experience in using the PercuSurge GuardWire Plus system as a distal protection device in patients with acute coronary syndrome and acute myocardial infarction. Forty-three patients received percutaneous coronary intervention with the GuardWire Plus system. Thirteen had unstable angina, five had non-Q myocardial infarction and 25 had ST segment elevation myocardial infarction. Forty-one target lesions were in native coronary vessels and two were in saphenous vein grafts. Total occlusion occurred in 18 patients. The mean occlusion time by the distal protective balloon was 262.8 +/- 114.1 s. Preoperatively, TIMI 0 flow was present in 18, TIMI II flow in two and TIMI III flow in 23 patients. Post-operatively, TIMI II and TIMI III flow were established in two and 41 patients, respectively. All procedures were successful and the GuardWire Plus system was successfully deployed in all but two patients. There was no procedure-related major adverse clinical event. There was no major adverse clinical event at 30 days. There was no device-related complication. We believe that the GuardWire Plus system is safe and feasible in patients with acute coronary syndrome and acute myocardial infarction.

Renal angioplasty under protection of the PercuSurge GuardWire Plus System.

Renal artery revascularization with angioplasty may preserve renal function and facilitate hypertension control. However, in a significant proportion of patients, renal function may deteriorate after the procedure. Distal microembolization has been implicated as a possible cause; it may be prevented with distal protection during the procedure. We report a case of successful renal angioplasty and stenting using the PercuSurge GuardWire Plus system.

Molecular features of human ubiquitin-like SUMO genes and their encoded proteins.

The SUMO (small ubiquitin-like modifier) protein and ubiquitin have similar 3-D structure. Sumolyzation and ubiquitination exhibit similar biological processes for post-translational modification. However, unlike ubiquitination, which targets proteins for degradation, sumolyzation participates in a number of cellular processes such as nuclear transport, transcriptional regulation, apoptosis and protein stability. The human genome contains three SUMO-1/2/3 functional genes, as well as eight SUMO-1 pseudogenes and 23 SUMO-2 pseudogenes, but no SUMO-3 pseudogenes. The protein-coding sequence of the SUMO-1 gene is interrupted by four introns, while those of SUMO-2 and SUMO-3 genes are interrupted by three introns. Human SUMO-1 protein exhibits 44% sequence identity with SUMO-2 and SUMO-3 proteins, while SUMO-2 and SUMO-3 proteins share 86% sequence identity. Phylogenetic analyses indicate that the SUMO-3 gene was derived from the SUMO-2 gene. SUMO-1 mRNA appears to be most abundant in human epithelial HeLa, kidney 293 and neuronal NT2 cells, while the SUMO-3 mRNA seems to be much less abundant than SUMO-2 mRNA, especially in HeLa and 293 cells. Many cellular proteins of high molecular weights were covalently modified by SUMO-1/2/3 proteins. However, some free form of SUMO-2/3 proteins was also detected. Most SUMO-1/2/3 proteins were shown to be localized on nuclear membrane, nuclear bodies and cytoplasm, respectively.