The Ubiquitin Ligase SMURF1 Catalyzes the Polyubiquitination of ADAR1 / 中国生物化学与分子生物学报

It is known that SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) mediates autophagy through its E3 ubiquitin ligase activity, but the ubiquitinated substrates of SMURF1 need to be further explored. In this paper, the interacting proteins of SMURF1 in THP-1 cells were captured and identified by co-immunoprecipitation (Co-IP) combined with mass spectrometry. It was found that SMURF1 could physically bind to 222 proteins in THP-1 cells, and Adenosine deaminase acting on RNA 1 (ADAR1) had a higher peptide binding score. SMURF1 overexpression vectors were constructed and transfected into HEK-293T cells, then Co-IP and Western blotting assays verified the interaction between exogenous SMURF1 and endogenous ADAR1. qRT-PCR and Western blotting assays were carried out after transfecting SMURF1 overexpression vectors in HEK-293T cells, which identified that overexpression of SMURF1 attenuated the protein levels of ADAR1 (P<0. 05). However, there was no significant difference in the mRNA level of ADAR1. HEK-293T cells with normal and overexpressing SMURF1 were treated with cycloheximide (CHX), respectively, and Western blotting assays showed a shortened half-life of ADAR1 after overexpression of SMURF1 (P < 0. 05). Furthermore, overexpression of SMURF1 increased the polyubiquitination level of ADAR1 as detected by Co-IP and Western blot (P<0. 05). After the proteasome inhibitor (MG132) treatment, the Western blotting assay was performed to demonstrate that the negative regulatory effect of SMURF1 on ADAR1 was weakened after the proteasome degradation pathway was attenuated (P<0. 05). This study shows that SMURF1 interacts with ADAR1, catalyzes the polyubiquitination of ADAR1 and mediates its degradation through the proteasome pathway, which provides a theoretical basis for exploring the various biological functions of SMURF1 by affecting the stability of ADAR1.

Effect of Smurf2 on Smad7, a negative regulator of TGF-β1 signaling pathway in hypertrophic scar and its mechanism / 中华整形外科杂志

Objective@#To investigate the effect and regulatory mechanism of Smurf2 on the negative regulator Smad7 of TGF-β1/Smad signaling pathway in hypertrophic scar fibroblasts.@*Methods@#From January to October 2014, 8 patients with hypertrophic scar after burn were admitted. The age of patients ranged from 1 year 8 months to 7 years, and the time of scar hyperplasia ranged from 2 to 12 months. The residual normal skin of the same patient was used as the control. The fibroblasts were isolated from hypertrophic scar and normal skin respectively and cultured. The third to fifth passage cells were used for the experiments. ① The protein expression of Smad7 in the two groups was detected by Western Blot. ② Hypertrophic scar fibroblasts and normal skin fibroblasts were treated with exogenous TGF-β1 at concentration of 10 ng/ml for 0 min, 5 min, 15 min, 30 min, 1 h, 2 h and 12 h. The expression of Smad7 protein and mRNA were detected by Western Blot and RT-PCR, respectively. ③ The cell lysates of the two groups were collected and incubated with the ubiquitin mixture for 1 h, 2 h and 6 h at 37℃, respectively. The degradation level of Smad7 protein was detected by Western Blot. ④ The cell lysate of hypertrophic scar fibroblasts was collected and incubated with ubiquitin mixture with or without proteasome inhibitor (MG132: MG115=1: 1) to study its inhibitory effect on the degradation of Smad7 in vitro. ⑤ Immunoprecipitation (IP) technique was used to detect the interaction between Smad 7 and E3 ubiquitin ligase Smurf2 in hypertrophic scar fibroblasts. ⑥ The expression of Smad7 protein in hypertrophic scar fibroblasts stimulated by TGF-β1 after Smurf2 silencing by small interference RNA (siRNA) technique were detected by Western blot.@*Results@#① There was no significant difference in the expression level of Smad7 protein between hypertrophic scar and normal skin fibroblasts(t=0.76, P=0.46). ② Expressions of Smad7 mRNA and protein in normal skin fibroblasts stimulated by exogenous TGF-β1 gradually increased in a time-dependent manner(P<0.05). The expression of Smad7 mRNA in the hypertrophic scar fibroblasts increased at all-time points except at 5min , (P<0.05), while there was no significant difference in the expression level of Smad7 protein at all-time points with or without TGF-β1 stimulation(P>0.05). ③ Degradation of Smad7 protein was enhanced in hypertrophic scar fibroblasts (the expression level of Smad 7 protein at each time point was compared with that of the control group and the last time point, P<0.05), while there was no significant difference in Smad7 protein degradation in normal skin fibroblasts(P=0.162). ④ Enhanced degradation of Smad7 in hypertrophic scar fibroblasts was blocked by the addition of the proteasome inhibitors MG132/MG115. ⑤ In hypertrophic scar fibroblasts, the Smurf2-Smad7 complex was detected, which indicated the interaction between Smurf2 and Smad7 in hypertrophic scar fibroblasts. ⑥ The expression of Smad7 protein was not increased in the hypertrophic scar fibroblasts stimulated by TGF-β1, whereas the stimulation of TGF-β1 increased the expression of Smad7 protein after silencing of Smurf2 gene expression.@*Conclusions@#In the hypertrophic scar fibroblasts, Smurf 2 attenuates the inhibitory effect of Smad 7 on TGF-β1 signaling pathway through the degradation of Smad7 by ubiquitination, which may be involved in the formation of hypertrophic scar.

Beta-Catenin Downregulation Contributes to Epidermal Growth Factor-induced Migration and Invasion of MDAMB231 Cells

We previously demonstrated that epidermal growth factor (EGF) enhances cell migration and invasion of breast cancer cells in a SMAD ubiquitination regulatory factor 1 (SMURF1)-dependent manner and that SMURF1 induces degradation of β-catenin in C2C12 cells. However, the relationship between EGF-induced SMURF1 and β-catenin expression in breast cancer cells remains unclear. So, we investigated if EGF and SMURF1 regulate β-catenin expression in MDAMB231 human breast cancer cells. When MDAMB231 cells were incubated with EGF for 24, 48, and 72 hours, EGF significantly increased expression levels of SMURF1 mRNA and protein while suppressing expression levels of β-catenin mRNA and protein. Overexpression of SMURF1 downregulated β-catenin mRNA and protein, whereas knockdown of SMURF1 increased β-catenin expression and blocked EGF-induced β-catenin downregulation. Knockdown of β-catenin enhanced cell migration and invasion of MDAMB231 cells, while β-catenin overexpression suppressed EGF-induced cell migration and invasion. Furthermore, knockdown of β-catenin enhanced vimentin expression and decreased cytokeratin expression, whereas β-catenin overexpression decreased vimentin expression and increased cytokeratin expression. These results suggest that EGF downregulates β-catenin in a SMURF1-dependent manner and that β-catenin downregulation contributes to EGF-induced cell migration and invasion in MDAMB breast cancer cells.

Ubiquitin ligase Smurf1 promotes poly-ubiquitination of PDK1 / 军事医学

Objective To investigate the mechanism of 3-phosphoinositide-dependent protein kinase 1(PDK1)poly-ubiquitination.Methods Co-immunoprecipitation(Co-IP)and Western blot(WB)were used to analyze poly-ubiquitination of PDK1.It was confirmed that ubiquitin ligase smad ubiquitylation regulatory factor 1(Smurf1)inprove PDK1 poly-ubiquitination within MEF cells,site-directed mutagenesis and WB before PDK1 poly-ubiquitination sites were determined.Results We found that PDK1 could undergoes poly-ubiquitination,ubiquitin ligase Smurf1 was found to be a direct E3 ligase for PDK1 poly-ubiquitination and might rely on the ubiquitin ligase Smurf 1 K699 site activity.K304 was PDK1 poly-ubiquitination modification site point.Conclusion The ubiquitin ligase Smurf1 can promate poly-ubiquitination of PDK1.

Establishment of two human tumor cell lines with lentivirus-mediated stably Smurf1 silencing and their effect on cell migration / 中国比较医学杂志

Objective To establish lentiviral expression vectors for Smurf1 silencing and assess the effects of Smurf1 silencing on cell migration.Methods HeLa and A549 cells were infected with lentiviral expression vectors for Smurf1 silencing respectively.After 7 days,the stable cell lines with Smurf1 silencing were obtained after puromycin-resistance screening,enrichment and expansion.The intracellular gene and protein levels of Smurf1 were detected by qPCR and western blot.Transwell assay was used to assess the effect of Smurf1 silencing on cell migration.Results The stable cell lines with Smurf1 silencing are constructed successfully.Silencing of Smurf1 down-regulated cell migration rate detected by Transwell assay.Conclusion Smurf1 promotes cell migration.

Ubiquitin ligase Smurf1 promotes Smurf2 neddylation modification / 军事医学

Objective To investigate the mechanism of Smad ubiquitination-related factor 2 (Smurf2)neddylation. Methods The Smurf2 protein level was tested by overexpression of Nedd8,while the method of immunoprecipitation(IP) and Western blotting were used to analyz Smurf2-Nedd8 modification.The GST-pulldown experiment was conducted to prove protein interactions.The protein was obtained by grinding mouse tissue and Western blotting was used to test the protein expression level.Results Over expression of Nedd8 could lead to the down regulation of the Smurf2′s protein level.Smurf1 and Smurf2 could interact in the GST-pulldown experiment. Smurf1 could enhance Smurf2-Nedd8 modification.The Smurf2′s protein level was up-regulated in mouse tissue of Smurf1 C426A.Conclusion There is some relationship and also difference between Smurf1 and Smurf2.Smurf1 can enhance Smurf2-Nedd8 modification.

Hepatocyte growth factor surpresses epithelial-mesenchymal transition through downregulating Smurf2 expression in rat NRK-52E cells / 中华肾脏病杂志

Objective To investigate the possible mechanism that hepatocyte growth factor (HGF) inhibits renal tubular epithelial-mesenchymal transition (EMT),and to determine whether Smurf2 expression induced by TGF-β1 can be reversed by HGF in normal rat kidney epithelial cells (NRK-52E).Methods Using rat NRK-52E cell line as an in vitro system,NRK-52E cells were incubated with 5 μg/L TGF-β1 for 0-24 h.Part of cells were pretreated with 20 μg/L HGF for 30 min or not,then incubated with or without 5 μg/L TGF-β1 for 1 h or 48 h.The other cells were transfected with pFlag-Smurf2 or Smurf2 siRNA for 24 h,then treated with or without 20 μg/L HGF for 24 h.The expressions of Smurf2,SnoN,E-cadherin,alpha-smooth muscle actin (α-SMA) and fibronectin (FN) were detected by Western blotting and indirect immunofluorescence staining assays.Results Compared to normal control,TGF-β1 could rapidly induce Smurf2 protein expression in a short time (P＜0.01).Meanwhile,the expressions of FN and α-SMA were significantly induced,and the expression of E-cadherin was reduced in NRK-52E cells by TGF-β1.In contrast,in the NRK-52E cells pretreated with HGF,HGF could obviously inhibit Smurf2 expression induced by TGF-β1,and reversed the down-regulation of SnoN (P＜0.01) and E-cadherin (P＜0.05),the up-regulation of α-SMA (P＜0.01) and FN (P＜0.01) induced by TGF-β1.Moreover,overexpression of Smurf2 in NRK-52E cells could partly inhibit the up-regulation of SnoN protein by HGF,while down-regulation of Smurf2 could up-regulate the expression of SnoN induced by HGF.Conclusions HGF can abolish EMT induced by TGF-β1 in renal tubular epithelial cells through down-regulating Smurf2 expression and suppressing ubiquitin-proteasome dependent degradation of SnoN.

Role of Smurf1 in bone morphogenetic protein-induced osteogenesis / 第二军医大学学报

Bone morphogenetic proteins (BMPs) are confirmed growth factors that can induce osteogenesis. They can bind to the surface receptors of target cells and activate a series of intracellular Smad proteins, which then transduct the signal into cell nuclear; and with the assistance of a series of cofactors, the osteogenic gene expression can be triggered. Endogenous Smurfl plays an inhibitory effect on the above process. This paper reviews the role of Smurf1 in bone morphogenetic protein-induced bone formation.

TGFbeta, activina e sinalização SMAD em câncer de tiróide / TGFbeta, activin and SMAD signalling in thyroid cancer

TGFbeta e activina são membros da superfamília TGFbeta e desempenham um amplo papel no desenvolvimento, proliferação e apoptose. Estes fatores de crescimento exercem seus efeitos biológicos ligando-se a receptores de membrana do tipo I e do tipo II que transduzem a sinalização até o núcleo através da fosforilação das proteínas R-SMADs (SMAD 2/3) e co-SMADs (SMAD4). O controle apropriado da via de TGFbeta/activina ainda depende da regulação negativa exercida pelo SMAD inibitório (SMAD7) e pelas enzimas E3 de ubiquitinação (Smurfs). Fisiologicamente, TGFbeta e activina atuam como potentes inibidores da proliferação na célula folicular tiroidiana. Desta forma, alterações de receptores e componentes da via de sinalização SMAD estão associadas a diferentes tipos de tumores. Desde que TGFbeta e activina geram sua sinalização intracelular utilizando os mesmos componentes da via SMAD, o desequilíbrio desta via prejudica dois processos anti-mitogênicos da célula. Nesta revisão, enfocamos aspectos que indicam o mecanismo de resistência ao efeito inibitório de TGFbeta e activina ocasionado pelo desequilíbrio da via de sinalização SMAD nas neoplasias da tiróide.

TGFbeta and activin are members of the TGFbeta superfamily and play a wide role in development, proliferation and apoptosis. These growth factors exert their biological effects by binding to the type I and II membrane receptors to transduce their signalling through the nucleus by phosphorylation of R-SMADs (SMAD 2/3) and co-SMADs (Smad 4). The proper control of TGFbeta/activin pathway is negatively regulated by inhibitory SMAD (SMAD7) and by E3 ubiquitination enzymes (Smurfs). Physiologically, TGFbeta and activin act as potent growth inhibitors in thyroid follicular cell. Thus, alterations in the receptors and components of SMAD signalling pathway are associated with several types of tumors. Since TGFbeta and activin generate their intracellular signalling through the same components of the SMAD pathway, the unbalance of this pathway impairs both of anti-mitogenic signals in the cell. This review addresses aspects of the molecular mechanisms in the understanding of resistance to the growth inhibitory effects of TGFbeta and activin due to the disequilibrium in the SMAD inhibitory pathway in thyroid neoplasia.

The Role of E3 Ubiquitin Ligase Smurfs in BMP and TGF-bata Signaling in Bone Cells / 生物化学与生物物理进展

The ubiquitin-proteasome system is composed by multiple enzymes which are ubiquitously expressed in mammalian cells and plays an essential role in a variety of biological processes. As key members of the protein degradation enzymatic system, the function of E3 ubiquitin ligases has been extensively investigated. BMP and TGF-? are critical molecules that regulate proliferation, differentiation and apoptosis of osteoblasts and chondrocytes through different signaling pathways. Resent findings indicate that the ubiquitin-proteasome system functions as a key regulator in bone cells and the E3 ligase mediates the proteolytic degradation of critical molecules in BMP and TGF-? signaling pathways. Recent progress on studies of HECT domain E3 ligase, Smurf, in osteoblasts and chondrocytes were summarized. The regulatory role of Smurf1 and Smurf2 in BMP and TGF-? signaling and osteoblast and chondrocyte function has been reviewed.

Expression of Smad 7,Smurf 1 and Smurf 2 in basal cen carcinoma and squamous cell carcinoma / 中华皮肤科杂志

Objective To investigate the expression and significance of Smad 7,Smurf 1 and Smurf 2 in basal cell carcinoma and squamous cell carcinoma.Methods Biopsy specimens were resected from 14 patients with basal cell carcinoma,19 patients with squamous cell carcinoma and 30 normal controls.Quanti- tative real-time PCR and immunohistochemical techniques were utilized to assess the expression of Smad 7, Smurf 1 and Smurf 2 in these specimens.Results The gray scale for staining of Smad 7,Smurf 1 and Smurf 2 was 166.61?7.11,166.08?8.71,and 166.25?8.15 respectively in basal cell carcinoma,161.66?5.52,166.84?9.27,and 169.98?9.48 respectively in squamous cell carcinoma.The expression levels of Smad 7,Smurf 1 and Smurf 2 were all significantly increased in basal cell carcinoma and squamous cell car- cinoma in comparison with normal controls.Conclusions The over-expression of Smad 7,Smurf 1 and Smurf 2 may interfere with transforming growth factor?signaling transduction pathway through several links,therefore prevent the inhibitory effect of transforming growth factor?on epidermal proliferation,and accelerate the abnormal proliferation in above epidermal tumors.