Sumoylation of p53 by the ubiquitin-like protein, SUMO-1/sentrin/PIC1, has been shown to stimulate its transcriptional activation activity. The SUMO E3 ligase, a key enzyme in the recognition of substrates to be sumoylated, has not yet been identified. We isolated PIAS1 (protein inhibitor of activated STAT1) as a SUMO-1 binding protein by yeast two-hybrid screening. In addition, PIAS1 bound p53 and Ubc9, the E2 for SUMO. PIAS1 that was mutated in the RING finger-like domain bound p53 and SUMO-1, but not Ubc9. PIAS1 catalyzed the sumoylation of p53 both in U2OS cells and in vitro in a domain-dependent manner. These data suggest that PIAS1 functions as a SUMO ligase, or possibly as a tightly bound regulator of it, toward p53.

Little is known about how synaptic activity is modulated in the central nervous system. We have identified SCRAPPER, a synapse-localized E3 ubiquitin ligase, which regulates neural transmission. SCRAPPER directly binds and ubiquitinates RIM1, a modulator of presynaptic plasticity. In neurons from Scrapper-knockout (SCR-KO) mice, RIM1 had a longer half-life with significant reduction in ubiquitination, indicating that SCRAPPER is the predominant ubiquitin ligase that mediates RIM1 degradation. As anticipated in a RIM1 degradation defect mutant, SCR-KO mice displayed altered electrophysiological synaptic activity, i.e., increased frequency of miniature excitatory postsynaptic currents. This phenotype of SCR-KO mice was phenocopied by RIM1 overexpression and could be rescued by re-expression of SCRAPPER or knockdown of RIM1. The acute effects of proteasome inhibitors, such as upregulation of RIM1 and the release probability, were blocked by the impairment of SCRAPPER. Thus, SCRAPPER has an essential function in regulating proteasome-mediated degradation of RIM1 required for synaptic tuning.

SUMO1/Smt3, a ubiquitin-like protein modifier, is known to conjugate to other proteins and modulate their functions in various important processes. Similar to the ubiquitin conjugation system, SUMO/Smt3 is transferred to substrate lysine residues through the thioester cascade of E1 (activating enzyme) and E2 (conjugating enzyme). In our previous report (Takahashi, Y., Toh-e, A., and Kikuchi, Y. (2001) Gene 275, 223-231), we showed that Siz1/Ull1 (YDR409w) of budding yeast, a member of the human PIAS family containing a RINGlike domain, is a strong candidate for SUMO1/Smt3 ligase because the SUMO1/Smt3 modification of septin components was abolished in the ull1 mutant and Ull1 associated with E2 (Ubc9) and the substrates (septin components) in immunoprecipitation experiments. Here we have developed an in vitro Smt3 conjugation system for a septin component (Cdc3) using purified recombinant proteins. In this system, Ull1 is additionally required as well as E1 (Sua1⅐Uba2 complex), E2 (Ubc9), and ATP. A cysteine residue of the RING-like domain was essential for the conjugation both in vivo and in vitro. Furthermore, a region containing the RING-like domain directly interacted with Ubc9 and Cdc3. Thus, this SUMO/Smt3 ligase functions as an adaptor between E2 and the target proteins.SUMO 1 (small ubiquitin-like modifier)/Smt3 is a member of a growing family of ubiquitin-related proteins and is known to be conjugated to RanGAP1, PML (promyelocytic leukemia), I B␣, p53, septins, etc. (1-4). Not only are the amino acid sequences and the three-dimensional structures similar between SUMO/ Smt3 and ubiquitin, but their conjugation systems and the enzymes involved are highly related (5-9). In the ubiquitin pathway a third enzyme, ubiquitin ligase (E3), is often required for the final transfer of this modifier and plays a crucial role by recognizing target proteins and by promoting their conjugation (10). It remains unknown, however, whether any SUMO1/Smt3 ligases (E3s) are involved in this conjugation pathway.In the ubiquitin pathway, some E3 components such as Apc11 of the anaphase-promoting complex and Rbx1 of the SCF (Skp1, cullin, F-box protein)⅐ubiquitin ligase complex contain a zinc-binding RING domain with an octet of ordered cysteine and histidine residues forming a cross-brace around two zinc atoms (11,12). This type of ubiquitin ligases (E3s) has to interact with E2 and the substrate at the same time because apparently they do not form the thioester bond with ubiquitin. In the case of c-Cbl proto-oncoprotein, its RING domain interacts with UbcH7 (E2), and the tyrosine kinase binding domain, a region close to the RING domain, recognizes its substrate. Thus, c-Cbl proto-oncoprotein functions as a bridging molecule between E2 and its substrate (13).In budding yeast, Smt3 is the only member of the SUMO family, and the Smt3 conjugation system is essential for mitotic growth. The lethality of the smt3 deletion mutant can be suppressed by expressing human SUMO1, suggesting that SUMO1 is a functional homologue of yeast Smt...

Objective-Statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) have pleiotropic vascular protective effects besides cholesterol lowering. Recently, experimental and clinical studies have indicated that senescence of endothelial cells is involved in endothelial dysfunction and atherogenesis. Therefore, the present study was performed to determine whether statins would reduce endothelial senescence and to clarify the molecular mechanisms underlying the antisenescent property of statins. Methods and Results-Senescent human umbilical vein endothelial cells were induced by hydrogen peroxide (H 2 O 2 ), as judged by senescence-associated ␤-galactosidase assay and cell morphological appearance. Atorvastatin, pravastatin, and pitavastatin inhibited the oxidative stress induced-endothelial senescence. These statins phosphorylated Akt at Ser473 and subsequently led to increased expression of endothelial nitric oxide synthase (eNOS), SIRT1, and catalase. Treatment with LY294002 or Akt short interfering RNA decreased the eNOS activation, SIRT1 expression, and antisenescent property of atorvastatin. Moreover, in streptozotocin-diabetic mice, administration of pitavastatin increased eNOS, SIRT1, and catalase expression and decreased endothelial senescence, but levels remained unaltered in Sirt1 knockout mice. Key Words: endothelium Ⅲ nitric oxide synthase Ⅲ SIRT1 Ⅲ senescence Ⅲ statin T he 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, statins, are effective in lowering the plasma concentration of low-density lipoprotein cholesterol and are widely used in patients with hypercholesterolemia. Recently, experimental and clinical evidence has indicated that the pleiotropic effects of statins involve improvement or restoration of endothelial function, enhanced activity of endothelial nitric oxide synthase (eNOS), and decreased oxidative stress. 1 Oxidative stress is implicated in the pathogenesis of cardiovascular diseases, such as atherosclerosis. 2 Excessive production of reactive oxygen species inflicts damage on endothelial cells and leads to the onset of endothelial senescence. Senescence of endothelial cells is involved in endothelial dysfunction and atherogenesis. 3 Histological study of human atherosclerotic lesions has demonstrated the existence of endothelial cells that exhibit the morphological features of senescence. 4 Assmus et al have shown that statins reduce senescence and increase proliferation of endothelial progenitor cells. 5 In Saccharomyces cerevisiae, the silent information regulator 2 (Sir2) family of genes governs budding exhaustion and replicative life span. 6,7 Sir2 has been identified as an NAD ϩ -dependent histone deacetylase and is responsible for maintenance of chromatin silencing and genome stability. 8 Sir2 genes are conserved during evolution, and 7 homologs of sirtuins (Sirt1 to Sirt7) have been cloned in mammals. Mammalian sirtuin 1 (Sirt1), the closest homolog of Sir2, regulates the cell cycle, senescence, apoptosis, and metabolism by interacting with a number of mo...

In immune cells, CD73 dephosphorylates and converts extracellular AMP into adenosine, which binds the A2A adenosine receptor (A2AR). Blockade of this interaction, which induces an immunosuppressed niche in the tumor microenvironment, represents a potential novel treatment strategy. The clinical significance of CD73 and A2AR expression in non-small-cell lung cancer (NSCLC), however, has yet to be thoroughly investigated. Here we evaluated CD73 and A2AR protein expression levels using immunohistochemistry in tissue microarrays containing 642 resected NSCLC specimens. Furthermore, we compared the expression profiles of 133 paired primary tumors and lymph node metastases. CD73 and A2AR expression levels were significantly higher in females than in males, in never smokers than in ever smokers, and in adenocarcinomas than in squamous cell carcinomas. Among adenocarcinomas, significantly higher CD73 and A2AR expression was observed in TTF-1-positive and mutant EGFR-positive tumors than in their counterparts. Compared with CD73, A2AR expression was more inconsistent between primary tumors and lymph node metastases. Among NSCLC patients, high CD73 expression was an independent indicator of poor prognosis in multivariate Cox regression analyses for overall survival [hazard ratio (HR), 2.18; 95% confidence interval (CI), 1.38–3.46] and recurrence-free survival (HR, 2.05; 95% CI, 1.42–2.95). In contrast, high A2AR expression was an independent predictor of favorable prognosis for overall survival (HR, 0.70; 95% CI, 0.50–0.98) and recurrence-free survival (HR, 0.74; 95% CI, 0.56–0.97). Together, these findings indicate that CD73 and A2AR have opposing prognostic effects, although cases involving CD73 or A2AR expression share some clinicopathological features.