SMURF2 predisposes cancer cell toward ferroptosis in GPX4-independent manners by promoting GSTP1 degradation.

Ferroptosis is a non-apoptotic form of regulated cell death. Glutathione (GSH) peroxidase 4 (GPX4) and GSH-independent ferroptosis suppressor protein 1 (FSP1) have been identified as major defenses. Here, we uncover a protective mechanism mediated by GSH S-transferase P1 (GSTP1) by monitoring proteinomic dynamics during ferroptosis. Dramatic downregulation of GSTP1 is caused by SMURF2-mediated GSTP1 ubiquitination and degradation at early stages of ferroptosis. Intriguingly, GSTP1 acts in GPX4- and FSP1-independent manners by catalyzing GSH conjugation of 4-hydroxynonenal and detoxifying lipid hydroperoxides via selenium-independent GSH peroxidase activity. Genetic modulation of the SMURF2/GSTP1 axis or the pharmacological inhibition of GSTP1's catalytic activity sensitized tumor responses to Food and Drug Administration (FDA)-approved ferroptosis-inducing drugs both in vitro and in vivo. GSTP1 expression also confers resistance to immune checkpoint inhibitors by blunting ferroptosis. Collectively, these findings demonstrate a GPX4/FSP1-independent cellular defense mechanism against ferroptosis and suggest that targeting SMURF2/GSTP1 to sensitize cancer cells to ferroptosis has potential as an anticancer therapy.

To Ub or not to Ub: a regulatory question in TGF-ß signaling.

The transforming growth factor ß (TGF-ß) superfamily controls a wide spectrum of biological processes in metazoans, including cell proliferation, apoptosis, differentiation, cell-fate determination, and embryonic development. Deregulation of TGF-ß-Smad signaling contributes to developmental anomalies and a variety of disorders and diseases such as tumorigenesis, fibrotic disorders, and immune diseases. In cancer, TGF-ß has dual effects through its antiproliferative and prometastatic actions. At the cellular level, TGF-ß functions mainly through the canonical Smad-dependent pathway in a cell type-specific and context-dependent manner. Accumulating evidence has demonstrated that ubiquitination plays a vital role in regulating TGF-ß-Smad signaling. We summarize current progress on ubiquitination (Ub) and the ubiquitin ligases that regulate TGF-ß-Smad signaling.

The E3 ligase SMURF1 stabilizes p27 via UbcH7 catalyzed K29-linked ubiquitin chains to promote cell migration SMURF1-UbcH7 K29 ubiquitination of p27 and cell migration.

Ubiquitination is a key regulator of protein stability and function. The multifunctional protein p27 is known to be degraded by the proteasome following K48-linked ubiquitination. However, we recently reported that when the ubiquitin-conjugating enzyme UbcH7 (UBE2L3) is overexpressed, p27 is stabilized, and cell cycle is arrested in multiple diverse cell types including eye lens, retina, HEK-293, and HELA cells. However, the ubiquitin ligase associated with this stabilization of p27 remained a mystery. Starting with an in vitro ubiquitination screen, we identified RSP5 as the yeast E3 ligase partner of UbcH7 in the ubiquitination of p27. Screening of the homologous human NEDD4 family of E3 ligases revealed that SMURF1 but not its close homolog SMURF2, stabilizes p27 in cells. We found that SMURF1 ubiquitinates p27 with K29O but not K29R or K63O ubiquitin in vitro, demonstrating a strong preference for K29 chain formation. Consistent with SMURF1/UbcH7 stabilization of p27, we also found that SMURF1, UbcH7, and p27 promote cell migration, whereas knockdown of SMURF1 or UbcH7 reduces cell migration. We further demonstrated the colocalization of SMURF1/p27 and UbcH7/p27 at the leading edge of migrating cells. In sum, these results indicate that SMURF1 and UbcH7 work together to produce K29-linked ubiquitin chains on p27, resulting in the stabilization of p27 and promoting its cell-cycle independent function of regulating cell migration.

Smurf1 polyubiquitinates on K285/K282 of the kinases Mst1/2 to attenuate their tumor-suppressor functions.

Sterile 20-like kinases Mst1 and Mst2 (Mst1/2) and large tumor suppressor 1/2 are core kinases to mediate Hippo signaling in maintaining tissue homeostasis. We have previously demonstrated that Smad ubiquitin (Ub) regulatory factor 1 (Smurf1), a HECT-type E3 ligase, ubiquitinates and in turn destabilizes large tumor suppressor 1/2 to induce the transcriptional output of Hippo signaling. Here, we unexpectedly find that Smurf1 interacts with and polyubiquitinates Mst1/2 by virtue of K27- and K29-linked Ub chains, resulting in the proteasomal degradation of Mst1/2 and attenuation of their tumor-suppressor functions. Among the potential Ub acceptor sites on Mst1/2, K285/K282 are conserved and essential for Smurf1-induced polyubiquitination and degradation of Mst1/2 as well as transcriptional output of Hippo signaling. As a result, K285R/K282R mutation of Mst1/2 not only negates the transcriptional output of Hippo signaling but enhances the tumor-suppressor functions of Mst1/2. Together, we demonstrate that Smurf1-mediated polyubiquitination on K285/K282 of Mst1/2 destabilizes Mst1/2 to attenuate their tumor-suppressor functions. Thus, the present study identifies Smurf1-mediated ubiquitination of Mst1/2 as a hitherto uncharacterized mechanism fine-tuning the Hippo signaling pathway and may provide additional targets for therapeutic intervention of diseases associated with this important pathway.

Hakai, a novel Runx2 interacting protein, augments osteoblast differentiation by rescuing Runx2 from Smurf2-mediated proteasome degradation.

Runt-related transcription factor 2 (Runx2) is a key regulator of osteoblast differentiation and bone formation. In Runx2-deficient embryos, skeletal development ceases at the cartilage anlage stage. These embryos die of respiratory failure upon birth and display a complete absence of bone and cartilage mineralization. Here, we identified Hakai, a type of E3 ubiquitin ligase as a potential Runx2 interacting partner through affinity pulldown-based proteomic approach. Subsequently, we observed that similar to Runx2, Hakai was downregulated in osteopenic ovariectomized rats, suggesting its involvement in bone formation. Consistent with this observation, Hakai overexpression significantly enhanced osteoblast differentiation in mesenchyme-like C3H10T1/2 as well as primary rat calvaria osteoblast (RCO) cells in vitro. Conversely, overexpression of a catalytically inactive Hakai mutant (C109A) exhibited minimal to no effect, whereas Hakai depletion markedly reduced endogenous Runx2 levels and impaired osteogenic differentiation in both C3H10T1/2 and RCOs. Mechanistically, Hakai physically interacts with Runx2 and enhances its protein turnover by rescuing it from Smad ubiquitination regulatory factor 2 (Smurf2)-mediated proteasome degradation. Wild-type Hakai but not Hakai-C109A inhibited Smurf2 protein levels through proteasome-mediated degradation. These findings underscore Hakai's functional role in bone formation, primarily through its positive modulation of Runx2 protein turnover by protecting it from Smurf2-mediated ubiquitin-proteasomal degradation. Collectively, our results demonstrate Hakai as a promising novel therapeutic target for osteoporosis.

The role of SMURFs in non-cancerous diseases.

The ubiquitin-proteasome system is a crucial mechanism for regulating protein levels in cells, with substrate-specific E3 ubiquitin ligases serving as an integral component of this system. Among these ligases are SMAD-specific E3 ubiquitin-protein ligase 1 (SMURF1) and SMAD-specific E3 ubiquitin-protein ligase 2 (SMURF2), which belong to the neural precursor cell-expressed developmentally downregulated 4 (NEDD4) subfamily of Homologous to E6-AP COOH terminus (HECT)-type E3 ligases. As E3 ligases, SMURFs have critical functions in regulating the stability of multiple proteins, thereby maintaining physiological processes such as cell migration, proliferation, and apoptosis. The occurrence of many diseases is attributed to abnormal cell physiology and an imbalance in cell homeostasis. It is noteworthy that SMURFs play pivotal roles in disease progression, with the regulatory functions being complex and either facilitative or inhibitory. In this review, we elucidate the mechanisms by which SMURF1 and SMURF2 can regulate disease progression in non-cancerous diseases. These significant findings offer potential novel therapeutic targets for various diseases and new avenues for research on SMURF proteins.

piR-368 promotes odontoblastic differentiation of dental papilla cells via the Smad1/5 signaling pathway by targeting Smurf1.

PURPOSE: The important role of non-coding RNAs in odontoblastic differentiation of dental tissue-derived stem cells has been widely demonstrated; however, whether piRNA (a subclass of non-coding RNA) involved in the course of odontoblastic differentiation is not yet available. This study aimed to investigate the expression profile of piRNA during odontogenic differentiation of mDPCs and the potential molecular mechanism in vitro. MATERIALS AND METHODS: The primary mouse dental papilla cells (mDPCs) were isolated from the first molars of 1-day postnatal Kunming mice. Then, they were cultured in odontogenic medium for 9 days. The expression profile of piRNA was detected by Small RNA sequencing. RT-qPCR was used to verify the elevation of piR-368. The mRNA and protein levels of mineralization markers were examined by qRT-PCR and Western blot analysis. Alkaline phosphatase (ALP) activity and alizarin red S staining were conducted to assess the odontoblastic differentiation ability. RESULTS: We validated piR-368 was significantly upregulated and interference with piR-368 markedly inhibited the odontogenic differentiation of mDPCs. In addition, the relationship between Smad1/5 signaling pathway and piR-368-induced odontoblastic differentiation has been discovered. Finally, we demonstrated Smurf1 as a target gene of piR-368 using dual-luciferase assays. CONCLUSION: This study was the first to illustrate the participation of piRNA in odontoblastic differentiation. We proved that piR-368 promoted odontoblastic differentiation of mouse dental papilla cells via the Smad1/5 signaling pathway by targeting Smurf1.

RUNX3 alleviates mitochondrial dysfunction and tubular damage by inhibiting TLR4/NF-κB signalling pathway in diabetic kidney disease.

AIM: The impaired function of tubular mitochondria is critical in diabetic kidney disease (DKD) progression. RUNX3 is down-regulated in DKD models. We intend to explore the effects of RUNX3 on mitochondrial dysfunction and renal tubule injury in DKD and related mechanisms. METHODS: The development of diabetes models involved injecting mice with streptozotocin while treating HK-2 cells with high glucose (HG). By using immunohistochemical techniques, the renal localizations of RUNX3 were identified. Levels of adenosine triphosphate (ATP), mitochondrial membrane potential, and biochemical index were detected by appropriate kits. Reactive oxygen species (ROS) generation was assessed with dihydroethidium and MitoSOX Red staining. Apoptosis was assessed by flow cytometry and TUNEL. RUNX3 ubiquitination was measured. RESULTS: RUNX3 was mainly present in renal tubules. Overexpressing RUNX3 increased Mfn2, Mfn1, ATP levels, and mitochondrial membrane potential, reduced Drp1 and ROS levels and cell apoptosis, as well as Cyt-C release into the cytoplasm. RUNX3 overexpression displayed a reduction in urinary albumin to creatinine ratio, Hemoglobin A1c, serum creatinine, and blood urea nitrogen. Overexpressing TLR4 attenuated the inhibitory effect of RUNX3 overexpression on mitochondrial dysfunction and cell apoptosis. HG promoted RUNX3 ubiquitination and SMURF2 expression. RUNX3 knockdown cancelled the inhibitory effect of SMURF2 on mitochondrial dysfunction and cell apoptosis. CONCLUSION: SMURF2 interference inhibits RUNX3 ubiquitination and TLR4/NF-κB signalling pathway, thereby alleviating renal tubule injury.

Citronellal improves endothelial dysfunction by affecting the stability of the GCH1 protein.

Endothelial dysfunction (ED) serves as the pathological basis for various cardiovascular diseases. Guanosine triphosphate cyclopyrrolone 1 (GCH1) emerges as a pivotal protein in sustaining nitric oxide (NO) production within endothelial cells, yet it undergoes degradation under oxidative stress, contributing to endothelial cell dysfunction. Citronellal (CT), a monoterpenoid, has been shown to ameliorate endothelial dysfunction induced by in atherosclerosis rats. However, whether CT can inhibit the degradation of GCH1 protein is not clear. It has been reported that ubiquitination may play a crucial role in regulating GCH1 protein levels and activities. However, the specific E3 ligase for GCH1 and the molecular mechanism of GCH1 ubiquitination remain unclear. Using data-base exploration analysis, we find that the levels of the E3 ligase Smad-ubiquitination regulatory factor 2 (Smurf2) negatively correlate with those of GCH1 in vascular tissues and HUVECs. We observe that Smurf2 interacts with GCH1 and promotes its degradation via the proteasome pathway. Interestingly, ectopic Smurf2 expression not only decreases GCH1 levels but also reduces cell proliferation and reactive oxygen species (ROS) levels, mostly because of increased GCH1 accumulation. Furthermore, we identify BH 4/eNOS as downstream of GCH1. Taken together, our results indicate that CT can obviously improve vascular endothelial injury in Type 1 diabetes mellitus (T1DM) rats and reverse the expressions of GCH1 and Smurf2 proteins in aorta of T1DM rats. Smurf2 promotes ubiquitination and degradation of GCH1 through proteasome pathway in HUVECs. We conclude that the Smurf2-GCH1 interaction might represent a potential target for improving endothelial injury.

TFPI2 suppresses the interaction of TGF-ß2 pathway regulators to promote endothelial-mesenchymal transition in diabetic nephropathy.

Endothelial-mesenchymal transition (EndMT) is an important source of myofibroblasts, but also contributes to the progression of diabetic nephropathy (DN). By several differential gene expression analyses from the Gene Expression Omnibus (GEO) database, the tissue factor pathway inhibitor 2 (TFPI2) gene, known as a tumor suppressor, was shown to be dysregulated in DN; however, the potential role and regulatory mechanism of TFPI2 in DN are unclear. Here, we found abnormal upregulation of TFPI2 in the renal cortex of diabetic mice, accompanied by impaired renal function. We also injected a single dose of adeno-associated virus (AAV)2 carrying shRNA targeting TFPI2 intravenously into these mice and found that knockdown of TFPI2 improved renal function and reduced renal fibrosis and cell apoptosis in experimental DN. Furthermore, hyperglycemia-induced EndMT was inhibited in the absence of TFPI2, as evidenced by increased expression of endothelial markers (VE-cadherin and CD31) and decreased expression of mesenchymal markers (α-SMA, desmin, and FSP-1). To further explore the mechanism in vitro, human renal glomerular endothelial cells (hRGECs) were incubated in the presence of high glucose or transforming growth factor beta (TGF-ß)2. TFPI2 deficiency inhibited high glucose-induced cell apoptosis and TGF-ß2-induced EndMT in hRGECs, while overexpression of TFPI2 had the opposite effects. Importantly, TGF-ß2 is a crucial driver of EndMT, and we found that TFPI2 promoted TGF-ß2/Smad signaling activation by interferring the interaction of TGF-ß pathway regulators (SMURF2 with SMAD7). Our results show that TFPI2 regulates EndMT and the TGF-ß2 signaling pathway and is a potential promoter of DN pathogenesis.

Transmembrane anterior posterior transformation 1 regulates BMP signaling and modulates the protein stability of SMAD1/5.

The bone morphogenetic protein (BMP) signaling pathway plays pivotal roles in various biological processes during embryogenesis and adult homeostasis. Transmembrane anterior posterior transformation 1 (TAPT1) is an evolutionarily conserved protein involved in murine axial skeletal patterning. Genetic defects in TAPT1 result in complex lethal osteochondrodysplasia. However, the specific cellular activity of TAPT1 is not clear. Herein, we report that TAPT1 inhibits BMP signaling and destabilizes the SMAD1/5 protein by facilitating its interaction with SMURF1 E3 ubiquitin ligase, which leads to SMAD1/5 proteasomal degradation. In addition, we found that the activation of BMP signaling facilitates the redistribution of TAPT1 and promotes its association with SMAD1. TAPT1-deficient murine C2C12 myoblasts or C3H/10T1/2 mesenchymal stem cells exhibit elevated SMAD1/5/9 protein levels, which amplifies BMP activation, in turn leading to a boost in the transdifferentiation or differentiation processing of these distinct TAPT1-deficient cell lines changing into mature osteoblasts. Furthermore, the enhancing effect of TAPT1 deficiency on osteogenic differentiation of C3H/10T1/2 cells was observed in an in vivo ectopic bone formation model. Importantly, a subset of TAPT1 mutations identified in humans with lethal skeletal dysplasia exhibited gain-of-function activity on SMAD1 protein levels. Thus, this finding elucidates the role of TAPT1 in the regulation of SMAD1/5 protein stability for controlling BMP signaling.

PTPN3 acts as a tumor suppressor and boosts TGF-ß signaling independent of its phosphatase activity.

TGF-ß controls a variety of cellular functions during development. Abnormal TGF-ß responses are commonly found in human diseases such as cancer, suggesting that TGF-ß signaling must be tightly regulated. Here, we report that protein tyrosine phosphatase non-receptor 3 (PTPN3) profoundly potentiates TGF-ß signaling independent of its phosphatase activity. PTPN3 stabilizes TGF-ß type I receptor (TßRI) through attenuating the interaction between Smurf2 and TßRI. Consequently, PTPN3 facilitates TGF-ß-induced R-Smad phosphorylation, transcriptional responses, and subsequent physiological responses. Importantly, the leucine-to-arginine substitution at amino acid residue 232 (L232R) of PTPN3, a frequent mutation found in intrahepatic cholangiocarcinoma (ICC), disables its role in enhancing TGF-ß signaling and abolishes its tumor-suppressive function. Our findings have revealed a vital role of PTPN3 in regulating TGF-ß signaling during normal physiology and pathogenesis.

Smurf1: A possible therapeutic target in dry age-related macular degeneration.

Smad ubiquitylation regulatory factor-1 (Smurf1) is one of C2-WW-HECT domain E3 ubiquitin ligases, it can regulate BMP pathway by mediating ubiquitylation degradation of Smad1/Smad5. Many functions about Smurf1 also are still unknown, especially in retina. This research is about to explore the role of Smurf1 in retina degeneration. Tail vein injection of sodium iodate (NaIO3) in C57BL/6J mice was the animal model of retina degeneration. In NaIO3 model, Smurf1 had more expression than normal mice. Specific Smurf1 inhibitor, A01, was injected into vitreous cavity. Results showed that inhibiting Smurf1 could alleviate acute retina injury, such as keeping a better retina structure in living imaging and histologic sections, less cell death and inflammation activation. Tert-butyl hydroperoxide (TBH) was used to establish oxidative stress injury in human retinal pigments epithelial cell line (ARPE-19). Oxidative stress injury gradually caused co-upregulation of Smurf1, TGF-ß1 and phosphorylated NF-κB (pNF-κB). TGF-ß1 could directly induce Smurf1 expression. Inhibiting Smurf1 had an anti-epithelial mesenchymal transition (anti-EMT) function. Similarly, A01 also could inhibit the expression of pNF-κB, NLRP3 and IL-1ß. At last, after searching bioinformatics database, Smurf1 had a possible interaction with beta-transducin repeat containing E3 ubiquitin protein ligase (ß-TrCP), another E3 ubiquitin ligases. ß-TrCP can mediate ubiquitination degradation of p-IκBα. Lentivirus-SMURF1 was used to overexpress Smurf1, and GS143 was used to inhibit ß-TrCP. The results showed Smurf1 could directly induce NF-κB, pNF-κB, and NLRP3 expression, and keep a stable ß-TrCP expression. However, inhibiting ß-TrCP could cause more NF-κB activation and NLRP3 expression. Therefore, ß-TrCP may play a negative role in NF-κB pathway activation. In summary, Smurf1 plays a role in exacerbating oxidative stress injury and inflammation in retina and may become a potential therapeutic target in ROS injury of retina.

The miR-15b-Smurf2-HSP27 axis promotes pulmonary fibrosis.

BACKGROUND: Heat shock protein 27 (HSP27) is overexpressed during pulmonary fibrosis (PF) and exacerbates PF; however, the upregulation of HSP27 during PF and the therapeutic strategy of HSP27 inhibition is not well elucidated. METHODS: We have developed a mouse model simulating clinical stereotactic body radiotherapy (SBRT) with focal irradiation and validated the induction of RIPF. HSP25 (murine form of HSP27) transgenic (TG) and LLC1-derived orthotropic lung tumor models were also used. Lung tissues of patients with RIPF and idiopathic pulmonary fibrosis, and lung tissues from various fibrotic mouse models, as well as appropriated cell line systems were used. Public available gene expression datasets were used for therapeutic response rate analysis. A synthetic small molecule HSP27 inhibitor, J2 was also used. RESULTS: HSP27 expression with its phosphorylated form (pHSP27) increased during PF. Decreased mRNA expression of SMAD-specific E3 ubiquitin-protein ligase 2 (Smurf2), which is involved in ubiquitin degradation of HSP27, was responsible for the increased expression of pHSP27. In addition, increased expression of miRNA15b was identified with decreased expression of Smurf2 mRNA in PF models. Inverse correlation between pHSP27 and Smurf2 was observed in the lung tissues of PF animals, an irradiated orthotropic lung cancer models, and PF tissues from patients. Moreover, a HSP27 inhibitor cross-linked with HSP27 protein to ameliorate PF, which was more effective when targeting the epithelial to mesenchymal transition (EMT) stage of PF. CONCLUSIONS: Our findings identify upregulation mechanisms of HSP27 during PF and provide a therapeutic strategy for HSP27 inhibition for overcoming PF.

HIF-1α-induced upregulated miR-322 forms a feedback loop by targeting Smurf2 and Smad7 to activate Smad3/ß-catenin/HIF-1α, thereby improving myocardial ischemia-reperfusion injury.

Myocardial ischemia/reperfusion injury (MIRI) is a major cause of heart failure after myocardial infarction. It has been reported that miR-322 is involved in MIRI progression, while the molecular mechanism of miR-322 in regulating MIRI progression needs to be further probed. MIRI cell model was established by oxygen-glucose deprivation/reoxygenation (OGD/R). Cell viability was assessed using MTS assay. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining were employed to analyze cell apoptosis. In addition, the interactions between miR-322, Smad7/Smurf2, hypoxia-inducible factor alpha (HIF-1α), and ß-catenin were verified by dual-luciferase reporter gene assay. Our results displayed that miR-322 was significantly downregulated in OGD/R-treated H9c2 cells, and its overexpression resulted in increased cell viability and reduced the apoptosis. Smurf2 and Smad7 were identified as the direct targets of miR-322. Smad7 knockdown or Smurf2 knockdown increased OGD/R-treated H9c2 cell viability and suppressed the apoptosis. Meanwhile, miR-322 mimics abolished the mitigating effect of Smad7 or Smurf2 overexpression on MIRI. In addition, the Smad3/ß-catenin pathway was identified as the downstream pathway of Smurf2/Smad7. Moreover, it was found that HIF-1α interacted with the miR-322 promoter, and ß-catenin interacted with the HIF-1α promoter to form a loop. HIF-1α-induced upregulated miR-322 activated the Smad3/ß-catenin pathway by targeting Smurf2 and Smad7 to improve MIRI; meanwhile, ß-catenin/HIF-1α formed a positive feedback loop to continuously improve MIRI.

The E3 ubiquitin ligase SMURF2 stabilizes RNA editase ADAR1p110 and promotes its adenosine-to-inosine (A-to-I) editing function.

Epitranscriptomic changes in RNA catalyzed by the RNA-editing enzyme ADAR1 play an essential role in the regulation of diverse molecular and cellular processes, both under physiological conditions and in disease states, including cancer. Yet, despite a growing body of evidence pointing to ADAR1 as a potential therapeutic target, the mechanisms regulating its cellular abundance and activity, particularly of its constitutively expressed and ubiquitous form, ADAR1p110, are poorly understood. Here, we report the HECT-type E3 ubiquitin ligase SMURF2 as a pivotal regulator of ADAR1p110. We show that SMURF2, which is primarily known to promote the ubiquitin-mediated degradation of its protein substrates, protects ADAR1p110 from proteolysis and promotes its A-to-I editase activity in human and mouse cells and tissues. ADAR1p110's interactome analysis performed in human cells also showed a positive influence of SMURF2 on the stability and function of ADAR1p110. Mechanistically, we found that SMURF2 directly binds, ubiquitinates and stabilizes ADAR1p110 in an E3 ubiquitin ligase-dependent manner, through ADAR1p110 ubiquitination at lysine-744 (K744). Mutation of this residue to arginine (K744R), which is also associated with several human disorders, including dyschromatosis symmetrica hereditaria (DSH) and some types of cancer, abolished SMURF2-mediated protection of ADAR1p110 from both proteasomal and lysosomal degradation and inactivated ADAR1p110-mediated RNA editing. Our findings reveal a novel mechanism underlying the regulation of ADAR1 in mammalian cells and suggest SMURF2 as a key cellular factor influencing the protein abundance, interactions and functions of ADAR1p110.

MiR-568 mitigated cardiomyocytes apoptosis, oxidative stress response and cardiac dysfunction via targeting SMURF2 in heart failure rats.

Chronic heart failure (CHF), a conventional, complex, and severe syndrome, is generally defined by myocardial output inadequate to satisfy the metabolic requirements of body tissues. Recently, miR-568 was identified to be down-regulated in CHF patients' sera and negatively correlated with left ventricular mass index in symptomatic CHF patients with systolic dysfunction. Nevertheless, the role of miR-568 during CHF development remains obscure. The current study is aimed to investigate the role of miR-568 in CHF. The MTT assay, flow cytometry analysis, RT-qPCR analysis, western blot analysis and luciferase reporter assays were conducted to figure out the function and potential mechanism of miR-568 in vitro. Rats were operated with aortic coarctation to establish CHF animal model. The effects of miR-568 and SMURF2 on CHF rats were evaluated by hematoxylin-eosin staining, Masson's staining, serum index testing, cardiac ultrasound detection, and TUNEL staining assays. We discovered that miR-568 level was downregulated by H2O2 treatment in cardiomyocytes. In mechanism, miR-568 directly targeted and negatively regulated SMURF2. In function, SMURF2 overexpression reversed the effects of miR-568 on cardiac function and histological changes in vivo. Additionally, SMURF2 overexpression reversed the effects of miR-568 on the content of LDH, AST, CK and CK-MB in vivo. Moreover, SMURF2 overexpression reversed the effects of miR-568 on oxidative stress response in vivo. MiR-568 mitigated cardiomyocytes apoptosis, oxidative stress response and cardiac dysfunction via targeting SMURF2 in CHF rats. This discovery may serve as a potential biomarker for CHF treatment.

Neddylation modification of the U3 snoRNA-binding protein RRP9 by Smurf1 promotes tumorigenesis.

Neddylation is a posttranslational modification that attaches ubiquitin-like protein Nedd8 to protein targets via Nedd8-specific E1-E2-E3 enzymes and modulates many important biological processes. Nedd8 attaches to a lysine residue of a substrate, not for degradation, but for modulation of substrate activity. We previously identified the HECT-type ubiquitin ligase Smurf1, which controls diverse cellular processes, is activated by Nedd8 through covalent neddylation. Smurf1 functions as a thioester bond-type Nedd8 ligase to catalyze its own neddylation. Numerous ubiquitination substrates of Smurf1 have been identified, but the neddylation substrates of Smurf1 remain unknown. Here, we show that Smurf1 interacts with RRP9, a core component of the U3 snoRNP complex, which is involved in pre-rRNA processing. Our in vivo and in vitro neddylation modification assays show that RRP9 is conjugated with Nedd8. RRP9 neddylation is catalyzed by Smurf1 and removed by the NEDP1 deneddylase. We identified Lys221 as a major neddylation site on RRP9. Deficiency of RRP9 neddylation inhibits pre-rRNA processing and leads to downregulation of ribosomal biogenesis. Consequently, functional studies suggest that ectopic expression of RRP9 promotes tumor cell proliferation, colony formation, and cell migration, whereas unneddylated RRP9, K221R mutant has no such effect. Furthermore, in human colorectal cancer, elevated expression of RRP9 and Smurf1 correlates with cancer progression. These results reveal that Smurf1 plays a multifaceted role in pre-rRNA processing by catalyzing RRP9 neddylation and shed new light on the oncogenic role of RRP9.

Smurf2 exerts neuroprotective effects on cerebral ischemic injury.

The present study aimed to explore specific mechanisms involved in mediating the neuroprotective effects of Smad ubiquitination regulatory factor 2 (Smurf2) in cerebral ischemic injury. A middle cerebral artery occlusion (MCAO) mouse model and an oxygen-glucose deprivation (OGD)-treated neuron model were developed. The expression of Smurf2, Yin Yang 1 (YY1), hypoxia-inducible factor-1 alpha (HIF1α), and DNA damage-inducible transcript 4 gene (DDIT4) was analyzed. Thereafter, the expression of Smurf2, YY1, HIF1α, and DDIT4 was altered in the MCAO mice and OGD-treated neurons. Apoptosis in tissues and cerebral infarction were assessed. In neurons, the expression of apoptosis-related proteins, viability, and apoptosis were assessed, followed by evaluation of lactate dehydrogenase leakage rate. The interaction between Smurf2 and YY1 was analyzed by coimmunoprecipitation assay and that between YY1 ubiquitination by in vivo ubiquitination experiment. The results showed downregulation of Smurf2 and upregulation of YY1, HIF1α, and DDIT4 in both MCAO mice and OGD-treated neurons. Smurf2 elevated YY1 ubiquitination and degradation, and YY1 increased HIF1α expression to promote DDIT4 in neurons. Overexpressed Smurf2 or downregulated YY1, HIF1α, or DDIT4 reduced the volume of cerebral infarction and apoptosis in MCAO mice, while enhancing cell viability and reducing apoptosis and lactate dehydrogenase leakage in OGD-treated neurons. In summary, our findings elucidated a neuroprotective role of Smurf2 in cerebral ischemic injury via inactivation of the YY1/HIF1α/DDIT4 axis.

Quantitative susceptibility mapping of the head-and-neck using SMURF fat-water imaging with chemical shift and relaxation rate corrections.

PURPOSE: To address the challenges posed by fat-water chemical shift artifacts and relaxation rate discrepancies to quantitative susceptibility mapping (QSM) outside the brain, and to generate accurate susceptibility maps of the head-and-neck at 3 and 7 Tesla. METHODS: Simultaneous Multiple Resonance Frequency (SMURF) imaging was extended to 7 Tesla and used to acquire head-and-neck gradient echo images at both 3 and 7 Tesla. Separated fat and water images were corrected for Type 1 (displacement) and Type 2 (phase discrepancy) chemical shift artefacts, and for the bias resulting from differences in T1 and T2∗ relaxation rates, recombined and used as the basis for QSM. A novel phase signal-based masking approach was used to generate head-and-neck masks. RESULTS: SMURF generated well-separated fat and water images of the head-and-neck. Corrections for chemical shift artefacts and relaxation rate differences removed overestimation of the susceptibility values, blurring in the susceptibility maps, and the disproportionate influence of fat in mixed voxels. The resulting susceptibility maps showed high correspondence between the paramagnetic areas and the locations of fatty tissues and the susceptibility estimates were similar to literature values. The proposed masking approach was shown to provide a simple means of generating head-and-neck masks. CONCLUSION: Corrections for Type 1 and Type 2 chemical shift artefacts and for fat-water relaxation rate differences, mainly in T1 , were shown to be required for accurate susceptibility mapping of fatty-body regions. SMURF made it possible to apply these corrections and generate high-quality susceptibility maps of the entire head-and-neck at both 3 and 7 Tesla.