Synoviolin, also called HRD1, is an E3 ubiquitin ligase and is implicated in endoplasmic reticulum -associated degradation. In mammals, Synoviolin plays crucial roles in various physiological and pathological processes, including embryogenesis and the pathogenesis of arthropathy. However, little is known about the molecular mechanisms of Synoviolin in these actions. To clarify these issues, we analyzed the profile of protein expression in synoviolinnull cells. Here, we report that Synoviolin targets tumor suppressor gene p53 for ubiquitination. Synoviolin sequestrated and metabolized p53 in the cytoplasm and negatively regulated its cellular level and biological functions, including transcription, cell cycle regulation and apoptosis. Furthermore, these p53 regulatory functions of Synoviolin were irrelevant to other E3 ubiquitin ligases for p53, such as MDM2, Pirh2 and Cop1, which form autoregulatory feedback loops. Our results provide novel insights into p53 signaling mediated by Synoviolin.

BackgroundHuman T-lymphotropic virus type 1 (HTLV-1) is a human retrovirus associated with both HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which is a chronic neuroinflammatory disease, and adult T-cell leukemia (ATL). The pathogenesis of HAM/TSP is known to be as follows: HTLV-1-infected T cells trigger a hyperimmune response leading to neuroinflammation. However, the HTLV-1-infected T cell subset that plays a major role in the accelerated immune response has not yet been identified.Principal FindingsHere, we demonstrate that CD4+CD25+CCR4+ T cells are the predominant viral reservoir, and their levels are increased in HAM/TSP patients. While CCR4 is known to be selectively expressed on T helper type 2 (Th2), Th17, and regulatory T (Treg) cells in healthy individuals, we demonstrate that IFN-γ production is extraordinarily increased and IL-4, IL-10, IL-17, and Foxp3 expression is decreased in the CD4+CD25+CCR4+ T cells of HAM/TSP patients as compared to those in healthy individuals, and the alteration in function is specific to this cell subtype. Notably, the frequency of IFN-γ-producing CD4+CD25+CCR4+Foxp3− T cells is dramatically increased in HAM/TSP patients, and this was found to be correlated with disease activity and severity.ConclusionsWe have defined a unique T cell subset—IFN-γ+CCR4+CD4+CD25+ T cells—that is abnormally increased and functionally altered in this retrovirus-associated inflammatory disorder of the central nervous system.

CBP and its homologue p300 play significant roles in cell differentiation, cell cycle, and anti-oncogenesis. We demonstrated that ␤-catenin, recently known as a potent oncogene, and CBP/p300 are associated through its CH3 region, which is a primary target of adenoviral oncoprotein E1A and various nuclear proteins, such as p53, cyclin E, and AP-1, and both are colocalized in the nuclear bodies. CBP/p300 potentiated Lef-mediated transactivation of ␤-catenin, and E1A, a potent inhibitor of CBP/p300, repressed its transactivation. Furthermore, overexpression of stable ␤-catenin mutant competitively suppressed the p53-dependent pathway. These may be a key mechanism of ␤-catenin involved in oncogenic events underlying disruption of tumor suppressor function through CBP/p300.The coactivator CBP/p300 family is a central regulator of gene expression (1-5) and important in cell differentiation, cell cycle, and anti-oncogenesis (6 -9). The importance of CBP/p300 in such cellular functions is supported by their ability to potentiate the activity of a large number of nuclear factors such as adenoviral oncoprotein E1A, p53, cyclin E, and AP-1 (10 -14). In addition, CBP/p300 has been proposed as a bridging protein, bringing together proteins of the RNA polymerase II-dependent basal transcription complex with either specific transcription factors or other cofactors.Drosophila Armadillo and its vertebrate homologue ␤-catenin are scaffold proteins in cadherin-mediated cell-cell adhesion, and they function as signal transducers to the nucleus in Wnt/Wingless pathway (15-17). ␤-Catenin translocated to the nucleus regulates expression of various downstream genes by acting as a coactivator of Lef/TCF family. Furthermore, recent studies suggested that stable mutants of ␤-catenin leading to accumulation in the nucleus are implicated in the initiation of some forms of colon cancers and melanomas (18 -21).The involvement of ␤-catenin in its nuclear functions, including transcriptional activation and oncogenic events, addresses the existence of bridging nuclear factors for recruitment of RNA polymerase II. To test this hypothesis, in the present study, we explored the possible association of ␤-catenin with CBP/p300 and attempted to define the biological function of CBP/p300 and ␤-catenin interaction. EXPERIMENTAL PROCEDURESCell Culture and Transfections and Reporter Gene Assays-HeLa cells, L cells, and HEK293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. HCT116 cells were maintained in McCoy 5A supplemented with 10% fetal bovine serum. Transfections were performed by the calcium phosphate method. Thirty ng of a RSV-Renilla luciferase control plasmid were included in each transfection experiment to control for the efficiency of transfection. To ensure equal DNA amounts, empty plasmids were added in each transfection. The luciferase activity was measured with AutoLumat (Berthold). The values were normalized to Renilla luciferase activity as an internal control.Antibodies-Anti-␤-catenin ...

No abstract

HNF-4, 1 a member of the steroid/thyroid nuclear receptor superfamily, is a transcriptional factor which expresses in the liver, intestine, kidney, and pancreatic ␤-cells (1, 2). It contains several functional domains: a ligand-independent activation domain (AF1), a zinc finger DNA binding domain, and a ligand-dependent activation domain (AF2) (3). HNF-4 binds to a specific DNA element as a homodimer and regulates the expression of many genes, involved in glucose, fatty acid, and cholesterol metabolisms (4 -6). The blood glucose levels are controlled by the balance of two opposing hormones, glucagon and insulin. Whereas glucagon decreases the activity of HNF-4, the effect of insulin on that of HNF-4 has not fully been understood yet (7-9).FKHR, a forkhead family member, is a transcriptional factor and regulates the expression of multiple genes, such as key enzymes of gluconeogenesis (10, 11). Insulin has a dynamic effect on the localization of FKHR, when phosphorylated by Akt at the three residues of FKHR: Thr-24, Ser-253, and Ser-316. Once phosphorylated, the cytoplasmic retention is induced, leading to inhibit the transcriptional activity. On the other hand, in the absence of insulin, FKHR is dephosphorylated and localized to the nucleus, where FKHR binds to the specific DNA element, resulting in transcriptional activation of the target genes (12-15).Recently, it has been reported that FKHR activates or represses the transactivation by nuclear receptor family members as a DNA binding-independent cofactor (16,17). In the present study, we analyzed the interaction of FKHR with HNF-4, the effect of FKHR on the transactivation mediated by HNF-4, and its molecular mechanism. FKHR associates with HNF-4 in vivo and in vitro and represses the transactivation by HNF-4 through the decrease in its DNA binding affinity. Interestingly, the inhibitory effect is canceled by insulin, resulting from the dissociation of HNF-4 from phosphorylated FKHR that subsequently translocates to the cytoplasm. This suggests the possibility that HNF-4 is a novel downstream target of insulin via FKHR as a signal-regulated transcriptional inhibitor. EXPERIMENTAL PROCEDURESCell Culture and Transfections and Reporter Gene Assays-HepG2 cells were cultured in Dalbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Transfections were performed by FuGENE-6 (Roche Molecular Biochemicals). Twenty ng of pCMV-␤gal plasmid were included in each transfection experiment to control for the efficiency of transfection. To ensure equal DNA amounts, empty plasmids were added in each transfection. The luciferase activity was measured with an ARVO™SX (Wallac Berthold). The values were normalized to ␤-galactosidase activity as an internal control.Plasmids-The hHNF-4 ␣2 cDNA was subcloned into pcDNA3 tagged with the HA epitope (pcDNA3HA). A series of HNF-4 deletion fragments were generated by PCR and subcloned into pGEX 4T-1 (Amersham Biosciences) or pcDNA3HA. The pGAL4-HNF-4 vector was made by subcloning hHNF-4 ␣2 cDNA tagged with the Gal4 DN...

Obesity is a major global public health problem, and understanding its pathogenesis is critical for identifying a cure. In this study, a gene knockout strategy was used in post-neonatal mice to delete synoviolin (Syvn)1/Hrd1/Der3, an ER-resident E3 ubiquitin ligase with known roles in homeostasis maintenance. Syvn1 deficiency resulted in weight loss and lower accumulation of white adipose tissue in otherwise wild-type animals as well as in genetically obese (ob/ob and db/db) and adipose tissue-specific knockout mice as compared to control animals. SYVN1 interacted with and ubiquitinated the thermogenic coactivator peroxisome proliferatoractivated receptor coactivator (PGC)-1b, and Syvn1 mutants showed upregulation of PGC-1b target genes and increase in mitochondrion number, respiration, and basal energy expenditure in adipose tissue relative to control animals. Moreover, the selective SYVN1 inhibitor LS-102 abolished the negative regulation of PGC-1b by SYVN1 and prevented weight gain in mice. Thus, SYVN1 is a novel post-translational regulator of PGC-1b and a potential therapeutic target in obesity treatment.

The EWS gene when fused to transcription factors such as the ETS family ATF-1, Wilms' tumor-1, and nuclear orphan receptors upon chromosomal translocation is thought to contribute the development of Ewing sarcoma and several malignant tumors. Although EWS is predicted to be an RNA-binding protein, an inherent EWS nuclear function has not yet been elucidated. In this study, we found that EWS associates with a transcriptional co-activator CREB-binding protein (CBP) and the hypophosphorylated RNA polymerase II, which are included preferentially in the transcription preinitiation complex. These interactions suggest the potential involvement of EWS in gene transcription, leading to the hypothesis that EWS may function as a co-activator of CBP-dependent transcription factors. Based on this hypothesis, we investigated the effect of EWS on the activation of nuclear receptors that are activated by CBP. Of nuclear receptors examined, hepatocyte nuclear factor 4-dependent transcription was selectively enhanced by EWS but not by an EWS mutant defective for CBP binding. These results suggest that EWS as a co-activator requires CBP for hepatocyte nuclear factor 4-mediated transcriptional activation.

BackgroundHuman T-lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a rare chronic neuroinflammatory disease. Since the disease course of HAM/TSP varies among patients, there is a dire need for biomarkers capable of predicting the rate of disease progression. However, there have been no studies to date that have compared the prognostic values of multiple potential biomarkers for HAM/TSP.Methodology/Principal FindingsPeripheral blood and cerebrospinal fluid (CSF) samples from HAM/TSP patients and HTLV-1-infected control subjects were obtained and tested retrospectively for several potential biomarkers, including chemokines and other cytokines, and nine optimal candidates were selected based on receiver operating characteristic (ROC) analysis. Next, we evaluated the relationship between these candidates and the rate of disease progression in HAM/TSP patients, beginning with a first cohort of 30 patients (Training Set) and proceeding to a second cohort of 23 patients (Test Set). We defined “deteriorating HAM/TSP” as distinctly worsening function (≥3 grades on Osame's Motor Disability Score (OMDS)) over four years and “stable HAM/TSP” as unchanged or only slightly worsened function (1 grade on OMDS) over four years, and we compared the levels of the candidate biomarkers in patients divided into these two groups. The CSF levels of chemokine (C-X-C motif) ligand 10 (CXCL10), CXCL9, and neopterin were well-correlated with disease progression, better even than HTLV-1 proviral load in PBMCs. Importantly, these results were validated using the Test Set.Conclusions/SignificanceAs the CSF levels of CXCL10, CXCL9, and neopterin were the most strongly correlated with rate of disease progression, they represent the most viable candidates for HAM/TSP prognostic biomarkers. The identification of effective prognostic biomarkers could lead to earlier detection of high-risk patients, more patient-specific treatment options, and more productive clinical trials.