Dataset Information

Inhibition of Cullin-RING E3 ubiquitin ligase 7 by simian virus 40 large T antigen.

ABSTRACT: Simian virus 40 (SV40) large tumor antigen (LT) triggers oncogenic transformation by inhibition of key tumor suppressor proteins, including p53 and members of the retinoblastoma family. In addition, SV40 transformation requires binding of LT to Cullin 7 (CUL7), a core component of Cullin-RING E3 ubiquitin ligase 7 (CRL7). However, the pathomechanistic effects of LT-CUL7 interaction are mostly unknown. Here we report both in vitro and in vivo experimental evidence that SV40 LT suppresses the ubiquitin ligase function of CRL7. We show that SV40 LT, but not a CUL7 binding-deficient mutant (LT(?69-83)), impaired 26S proteasome-dependent proteolysis of the CRL7 target protein insulin receptor substrate 1 (IRS1), a component of the insulin and insulin-like growth factor 1 signaling pathway. SV40 LT expression resulted in the accumulation and prolonged half-life of IRS1. In vitro, purified SV40 LT reduced CRL7-dependent IRS1 ubiquitination in a concentration-dependent manner. Expression of SV40 LT, or depletion of CUL7 by RNA interference, resulted in the enhanced activation of IRS1 downstream signaling pathways phosphatidylinositol-3-kinase/AKT and Erk mitogen-activated pathway kinase, as well as up-regulation of the downstream target gene c-fos. Finally, SV40 LT-positive carcinoma of carcinoembryonic antigen 424/SV40 LT transgenic mice displayed elevated IRS1 protein levels and activation of downstream signaling. Taken together, these data suggest that SV40 LT protects IRS1 from CRL7-mediated degradation, thereby sustaining high levels of promitogenic IRS1 downstream signaling pathways.

SUBMITTER: Hartmann T

PROVIDER: S-EPMC3948260 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Inhibition of Cullin-RING E3 ubiquitin ligase 7 by simian virus 40 large T antigen.

Hartmann Thomas T Xu Xinsong X Kronast Mira M Muehlich Susanne S Meyer Kathleen K Zimmermann Wolfgang W Hurwitz Jerard J Pan Zhen-Qiang ZQ Engelhardt Stefan S Sarikas Antonio A

Proceedings of the National Academy of Sciences of the United States of America 20140218 9

Simian virus 40 (SV40) large tumor antigen (LT) triggers oncogenic transformation by inhibition of key tumor suppressor proteins, including p53 and members of the retinoblastoma family. In addition, SV40 transformation requires binding of LT to Cullin 7 (CUL7), a core component of Cullin-RING E3 ubiquitin ligase 7 (CRL7). However, the pathomechanistic effects of LT-CUL7 interaction are mostly unknown. Here we report both in vitro and in vivo experimental evidence that SV40 LT suppresses the ubiq ...[more]

PMID: 24550499

Similar Datasets

Project description:Fibrosis is a pathognomonic feature of structural heart disease and counteracted by distinct cardioprotective mechanisms, e.g. activation of the phosphoinositide 3-kinase (PI3K) / AKT pro-survival pathway. The Cullin-RING E3 ubiquitin ligase 7 (CRL7) was identified as negative regulator of PI3K/AKT signalling in skeletal muscle, but its role in the heart remains to be elucidated. Here, we sought to determine whether CRL7 modulates to cardiac fibrosis following pressure overload and dissect its underlying mechanisms. For inactivation of CRL7, the Cullin 7 (Cul7) gene was deleted in cardiac myocytes (CM) by injection of adeno-associated virus subtype 9 (AAV9) vectors encoding codon improved Cre-recombinase (AAV9-CMV-iCre) in Cul7flox/flox mice. In addition, Myosin Heavy Chain 6 (Myh6; alpha-MHC)-MerCreMer transgenic mice with tamoxifen-induced CM-specific expression of iCre were used as alternate model. After transverse aortic constriction (TAC), causing chronic pressure overload and fibrosis, AAV9-CMV-iCre induced Cul7-/- mice displayed a ~50% reduction of interstitial cardiac fibrosis when compared to Cul7+/+ animals (6.7% vs. 3.4%, p<0.01). Similar results were obtained with Cul7flox/flox Myh6-Mer-Cre-MerTg(1/0) mice which displayed a ~30% reduction of cardiac fibrosis after TAC when compared to Cul7+/+ Myh6-Mer-Cre-MerTg(1/0) controls after TAC surgery (12.4% vs. 8.7%, p<0.05). No hemodynamic alterations were observed. AKTSer473 phosphorylation was increased 3-fold (p<0.01) in Cul7-/- vs. control mice, together with a ~78% (p<0.001) reduction of TUNEL-positive apoptotic cells three weeks after TAC. In addition, CM-specific expression of a dominant-negative CUL71152stop mutant resulted in a 16.3-fold decrease (p<0.001) of in situ end-labelling (ISEL) positive apoptotic cells. Collectively, our data demonstrate that CM-specific ablation of Cul7 restrains myocardial fibrosis and apoptosis upon pressure overload, and introduce CRL7 as a potential target for anti-fibrotic therapeutic strategies of the heart.

Project description:UnlabelledThe rotavirus nonstructural protein NSP1 acts as an antagonist of the host antiviral response by inducing degradation of key proteins required to activate interferon (IFN) production. Protein degradation induced by NSP1 is dependent on the proteasome, and the presence of a RING domain near the N terminus has led to the hypothesis that NSP1 is an E3 ubiquitin ligase. To examine this hypothesis, pulldown assays were performed, followed by mass spectrometry to identify components of the host ubiquitination machinery that associate with NSP1. Multiple components of cullin RING ligases (CRLs), which are essential multisubunit ubiquitination complexes, were identified in association with NSP1. The mass spectrometry was validated in both transfected and infected cells to show that the NSP1 proteins from different strains of rotavirus associated with key components of CRL complexes, most notably the cullin scaffolding proteins Cul3 and Cul1. In vitro binding assays using purified proteins confirmed that NSP1 specifically interacted with Cul3 and that the N-terminal region of Cul3 was responsible for binding to NSP1. To test if NSP1 used CRL3 to induce degradation of the target protein IRF3 or β-TrCP, Cul3 levels were knocked down using a small interfering RNA (siRNA) approach. Unexpectedly, loss of Cul3 did not rescue IRF3 or β-TrCP from degradation in infected cells. The results indicate that, rather than actively using CRL complexes to induce degradation of target proteins required for IFN production, NSP1 may use cullin-containing complexes to prevent another cellular activity.ImportanceThe ubiquitin-proteasome pathway plays an important regulatory role in numerous cellular functions, and many viruses have evolved mechanisms to exploit or manipulate this pathway to enhance replication and spread. Rotavirus, a major cause of severe gastroenteritis in young children that causes approximately 420,000 deaths worldwide each year, utilizes the ubiquitin-proteasome system to subvert the host innate immune response by inducing the degradation of key components required for the production of interferon (IFN). Here, we show that NSP1 proteins from different rotavirus strains associate with the scaffolding proteins Cul1 and Cul3 of CRL ubiquitin ligase complexes. Nonetheless, knockdown of Cul1 and Cul3 suggests that NSP1 induces the degradation of some target proteins independently of its association with CRL complexes, stressing a need to further investigate the mechanistic details of how NSP1 subverts the host IFN response.

Dataset Information

Inhibition of Cullin-RING E3 ubiquitin ligase 7 by simian virus 40 large T antigen.

Publications

Inhibition of Cullin-RING E3 ubiquitin ligase 7 by simian virus 40 large T antigen.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets