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1.
Chem Biol Interact ; 337: 109394, 2021 Mar 01.
Article in English | MEDLINE | ID: mdl-33508304

ABSTRACT

The imbalance of SUMOylation is related to different cancers, including gastric cancer (GC). Ginkgolic acid (GA) inhibits the growth and invasion of many cancer cells, and it has been reported to restrain SUMOylation. However, the role of GA in GC and whether it functions through SUMOylation remains to be clarified. Our research revealed that GA (15:1) inhibited cell proliferation, migration, epithelial-mesenchymal transition (EMT) and overall protein SUMOylation in BGC823 and HGC27 cells. In addition, knockdown of SUMO1 (small ubiquitin-like modifier) instead of SUMO2/3 played a similar role to GA in cell behaviors. Besides, nuclear IGF-1R (insulin-like growth factor 1 receptor) expression was markedly upregulated in GC cells compared to normal gastric epithelial cells. GA prevented IGF-1R from binding to SUMO1, thereby suppressing its nuclear accumulation. Further research found that IGF-1R directly bound to SNAI2 (snail family zinc finger 2) promoter. The interference of IGF-1R downregulated the mRNA and protein levels of SNAI2, while the overexpression of SUMO1, IGF-1R and UBC9 (SUMO-conjugating enzyme) played the opposite role. Furthermore, the co-transfection of SUMO1, UBC9 and IGF-1R vectors or the overexpression of SNAI2 reversed the inhibitory effects of GA on cell proliferation, migration and EMT. Finally, GA impeded the growth of GC xenografts and decreased the expression of nuclear IGF-1R and SNAI2 in vivo. In conclusion, these findings demonstrated that GA hindered the progression of GC by inhibiting the SUMOylation of IGF-1R. Thus, GA might be a promising therapeutic for GC.


Subject(s)
Cell Proliferation/drug effects , Epithelial-Mesenchymal Transition/drug effects , Receptor, IGF Type 1/metabolism , Salicylates/pharmacology , Animals , Cell Line, Tumor , Cell Movement/drug effects , Female , Gene Expression Regulation/drug effects , Humans , Mice , Mice, Nude , RNA Interference , RNA, Small Interfering/metabolism , Receptor, IGF Type 1/antagonists & inhibitors , Receptor, IGF Type 1/genetics , SUMO-1 Protein/antagonists & inhibitors , SUMO-1 Protein/genetics , SUMO-1 Protein/metabolism , Salicylates/therapeutic use , Small Ubiquitin-Related Modifier Proteins/antagonists & inhibitors , Small Ubiquitin-Related Modifier Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/metabolism , Snail Family Transcription Factors/genetics , Snail Family Transcription Factors/metabolism , Stomach Neoplasms/drug therapy , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , Sumoylation/drug effects , Transplantation, Heterologous
2.
Br J Cancer ; 124(3): 531-538, 2021 02.
Article in English | MEDLINE | ID: mdl-33071285

ABSTRACT

An urgent medical need to develop novel treatment strategies for patients with pancreatic ductal adenocarcinoma (PDAC) exists. However, despite various efforts in the histopathological and molecular subtyping of PDAC, novel targeted or specific therapies have not been established. Posttranslational modifications (PTMs) with ubiquitin-like proteins, including small ubiquitin-like modifiers (SUMOs), mediate numerous processes that can contribute to the fitness and survival of cancer cells. The contribution of SUMOylation to transcriptional control, DNA repair pathways, mitotic progression, and oncogenic signalling has been described. Here we review functions of the SUMO pathway in PDAC, with a special focus on its connection to an aggressive subtype of the disease characterised by high MYC activity, and discuss SUMOylation inhibitors under development for precise PDAC therapies.


Subject(s)
Carcinoma, Pancreatic Ductal/metabolism , Pancreatic Neoplasms/metabolism , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-myc/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Survival/physiology , Cysteine Endopeptidases/drug effects , Cysteine Endopeptidases/metabolism , DNA Repair/physiology , Enzyme Inhibitors/pharmacology , Humans , Mitosis/physiology , Pancreatic Neoplasms/pathology , Poly-ADP-Ribose Binding Proteins/metabolism , Promyelocytic Leukemia Protein/metabolism , Protein Inhibitors of Activated STAT/metabolism , Proto-Oncogene Proteins c-myc/genetics , SUMO-1 Protein/antagonists & inhibitors , SUMO-1 Protein/metabolism , Signal Transduction/physiology , Small Ubiquitin-Related Modifier Proteins/antagonists & inhibitors , Sumoylation/drug effects , Synthetic Lethal Mutations , Ubiquitin-Conjugating Enzymes/antagonists & inhibitors , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitins/antagonists & inhibitors , Ubiquitins/metabolism
3.
Int J Oncol ; 57(3): 780-790, 2020 09.
Article in English | MEDLINE | ID: mdl-32705164

ABSTRACT

It has been controversial whether patients with hepatocellular carcinoma (HCC) should receive glucocorticoid therapy during chemotherapy. Recent studies have demonstrated that glucocorticoids increase the therapeutic sensitivity of tumors to some chemotherapeutic drugs, but the specific mechanism remains unclear. In the present study, dexamethasone (Dex) was used to treat HCC stem cells. The results demonstrated that Dex reduced stemness maintenance and self­renewal of HCC stem cells, promoted epithelial­to­mesenchymal transition, inhibited migration and angiogenesis and, more importantly, increased cell sensitivity to the herpes simplex virus thymidine kinase/ganciclovir drug system in vitro and in vivo. Further mechanistic analyses demonstrated that Dex inhibited small ubiquitin­like modifier (SUMO) modification of several proteins in HCC stem cells, including hypoxia­inducible factor (HIF)­1α, an important hypoxia tolerance protein, and octamer­binding transcription factor 4 (Oct4), a crucial stemness maintenance protein. Inducing deSUMOylation of HIF­1α and Oct4 reduced their accumulation in the nucleus, thereby inhibiting tumor angiogenesis and stemness maintenance. These findings provide a new perspective to the study of the mechanism underlying the anti­hepatocarcinogenesis effects of Dex. Due to the few side effects of glucocorticoids at low doses and their anti­inflammatory effects, the appropriate combination of glucocorticoids and chemotherapeutic drugs is expected to improve the survival of HCC patients and their prognosis.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Carcinoma, Hepatocellular/drug therapy , Dexamethasone/pharmacology , Liver Neoplasms/drug therapy , Neoplastic Stem Cells/drug effects , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Dexamethasone/therapeutic use , Drug Resistance, Neoplasm/drug effects , Drug Synergism , Epithelial-Mesenchymal Transition/drug effects , Female , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Liver Neoplasms/pathology , Mesenchymal Stem Cells , Mice , Neoplastic Stem Cells/pathology , Octamer Transcription Factor-3/metabolism , SUMO-1 Protein/antagonists & inhibitors , SUMO-1 Protein/metabolism , Signal Transduction , Spheroids, Cellular , Sumoylation/drug effects , Xenograft Model Antitumor Assays
4.
Biochem Pharmacol ; 178: 114066, 2020 08.
Article in English | MEDLINE | ID: mdl-32502496

ABSTRACT

Oxidative stress is a toxic cellular condition, strictly related to inflammation and known to be a common feature of many neurodegenerative diseases. The imbalanced redox state modifies several molecular processes including protein SUMOylation, JNK and Tau protein activation, important actors in Alzheimer's disease. In this study, we showed a strong interaction among SUMO-1-JNK-Tau proteins and their molecular targets in an in vitro model (SHSY5Y cell line) of oxidative stress in which a significant reduction of cell viability and an augmented cell death was induced by increased doses of H2O2. The evoked oxidative stress led to a deficiency in the degradation system showing altered levels of Caspase-3, LC3BII/I and Ubiquitin. Curcumin, a natural compound with anti-oxidant and anti-inflammatory effects, demonstrated to tackle oxidative stress re-equilibrating SUMO-1, JNK and Tau functions. Importantly, 5 µM of curcumin induced an efficient recovery of cell viability, a reduction of cell death and a normalization of altered protein degradation marker levels. Interestingly, we found that H2O2 treatment induced a strong co-localization of SUMO-1-p-JNK-Tau proteins in nuclear bodies (NBs) and that curcumin was able to reduce these nuclear aggregates. These results highlight the SUMO-1-JNK-Tau axis key role in oxidative stress and the protective effect of curcumin against this pathological event, focusing on the importance of SUMO/deSUMOylation balance to regulate essential cellular processes.


Subject(s)
Curcumin/pharmacology , JNK Mitogen-Activated Protein Kinases/metabolism , Oxidative Stress/drug effects , SUMO-1 Protein/metabolism , tau Proteins/metabolism , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antioxidants/pharmacology , Cell Line, Tumor , Dose-Response Relationship, Drug , Humans , Hydrogen Peroxide/toxicity , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , Oxidative Stress/physiology , Protective Agents/pharmacology , SUMO-1 Protein/antagonists & inhibitors , Sumoylation/drug effects , Sumoylation/physiology , tau Proteins/antagonists & inhibitors
5.
Adv Exp Med Biol ; 1233: 29-54, 2020.
Article in English | MEDLINE | ID: mdl-32274752

ABSTRACT

Ubiquitin defines a family of approximately 20 peptidic posttranslational modifiers collectively called the Ubiquitin-like (UbLs). They are conjugated to thousands of proteins, modifying their function and fate in many ways. Dysregulation of these modifications has been implicated in a variety of pathologies, in particular cancer. Ubiquitin, SUMO (-1 to -3), and Nedd8 are the best-characterized UbLs. They have been involved in the regulation of the activity and/or the stability of diverse components of various oncogenic or tumor suppressor pathways. Moreover, the dysregulation of enzymes responsible for their conjugation/deconjugation has also been associated with tumorigenesis and cancer resistance to therapies. The UbL system therefore constitutes an attractive target for developing novel anticancer therapeutic strategies. Here, we review the roles and dysregulations of Ubiquitin, SUMO, and Nedd8 pathways in tumorigenesis, as well as recent advances in the identification of small molecules targeting their conjugating machineries for potential application in the fight against cancer.


Subject(s)
Molecular Targeted Therapy , NEDD8 Protein/antagonists & inhibitors , Neoplasms/drug therapy , Neoplasms/metabolism , SUMO-1 Protein/antagonists & inhibitors , Ubiquitin/antagonists & inhibitors , Genes, Tumor Suppressor , Humans , Neoplasms/genetics
6.
Oxid Med Cell Longev ; 2019: 1232146, 2019.
Article in English | MEDLINE | ID: mdl-31428220

ABSTRACT

BACKGROUND: Zinc plays a role in mitophagy and protects cardiomyocytes from ischemia/reperfusion injury. This study is aimed at investigating whether SUMOylation of Drp1 is involved in the protection of zinc ion on cardiac I/R injury. METHODS: Mouse hearts were subjected to 30 minutes of regional ischemia followed by 2 hours of reperfusion (ischemia/reoxygenation (I/R)). Infarct size and apoptosis were assessed. HL-1 cells were subjected to 24 hours of hypoxia and 6 hours of reoxygenation (hypoxia/reoxygenation (H/R)). Zinc was given 5 min before reperfusion for 30 min. SENP2 overexpression plasmid (Flag-SENP2), Drp1 mutation plasmid (Myc-Drp1 4KR), and SUMO1 siRNA were transfected into HL-1 cells for 48 h before hypoxia. Effects of zinc on SUMO family members were analyzed by Western blotting. SUMOylation of Drp1, apoptosis and the collapse of mitochondrial membrane potential (ΔΨm), and mitophagy were evaluated. RESULTS: Compared with the control, SUMO1 modification level of proteins in the H/R decreased, while this effect was reversed by zinc. In the setting of H/R, zinc attenuated myocardial apoptosis, which was reversed by SUMO1 siRNA. Similar effects were observed in SUMO1 KO mice exposed to H/R. In addition, the dynamin-related protein 1 (Drp1) is a target protein of SUMO1. The SUMOylation of Drp1 induced by zinc regulated mitophagy and contributed to the protective effect of zinc on H/R injury. CONCLUSIONS: SUMOylation of Drp1 played an essential role in zinc-induced cardio protection against I/R injury. Our findings provide a promising therapeutic approach for acute myocardial I/R injury.


Subject(s)
Dynamins/metabolism , Myocardial Reperfusion Injury/pathology , Protective Agents/pharmacology , Zinc/pharmacology , Animals , Apoptosis/drug effects , Cell Hypoxia , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Dynamins/genetics , Heart/drug effects , Membrane Potential, Mitochondrial/drug effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutagenesis , Myocardial Reperfusion Injury/prevention & control , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Protective Agents/therapeutic use , RNA Interference , RNA, Small Interfering/metabolism , Reactive Oxygen Species/metabolism , SUMO-1 Protein/antagonists & inhibitors , SUMO-1 Protein/genetics , SUMO-1 Protein/metabolism , Sumoylation/drug effects
7.
Int J Mol Sci ; 20(2)2019 Jan 17.
Article in English | MEDLINE | ID: mdl-30658479

ABSTRACT

Post-translational modifications of host or viral proteins are key strategies exploited by viruses to support virus replication and counteract host immune response. SUMOylation is a post-translational modification process mediated by a family of ubiquitin-like proteins called small ubiquitin-like modifier (SUMO) proteins. Multiple sequence alignment of 78 representative flaviviruses showed that most (72/78, 92.3%) have a putative SUMO-interacting motif (SIM) at their non-structural 5 (NS5) protein's N-terminal domain. The putative SIM was highly conserved among 414 pre-epidemic and epidemic Zika virus (ZIKV) strains, with all of them having a putative SIM core amino acid sequence of VIDL (327/414, 79.0%) or VVDL (87/414, 21.0%). Molecular docking predicted that the hydrophobic SIM core residues bind to the ß2 strand of the SUMO-1 protein, and the acidic residues flanking the core strengthen the binding through interactions with the basic surface of the SUMO protein. The SUMO inhibitor 2-D08 significantly reduced replication of flaviviruses and protected cells against ZIKV-induced cytopathic effects in vitro. A SIM-mutated ZIKV NS5 failed to efficiently suppress type I interferon signaling. Overall, these findings may suggest SUMO modification of the viral NS5 protein to be an evolutionarily conserved post-translational modification process among flaviviruses to enhance virus replication and suppress host antiviral response.


Subject(s)
Viral Nonstructural Proteins/metabolism , Zika Virus Infection/virology , Zika Virus/physiology , Amino Acid Sequence , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Conserved Sequence , Humans , Interferon Type I/metabolism , Models, Molecular , Phylogeny , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Protein Processing, Post-Translational/drug effects , SUMO-1 Protein/antagonists & inhibitors , SUMO-1 Protein/chemistry , SUMO-1 Protein/metabolism , Signal Transduction , Structure-Activity Relationship , Sumoylation/drug effects , Viral Nonstructural Proteins/chemistry , Virus Replication/drug effects , Zika Virus/classification , Zika Virus/drug effects , Zika Virus Infection/metabolism
8.
Thorac Cancer ; 10(1): 33-40, 2019 01.
Article in English | MEDLINE | ID: mdl-30393970

ABSTRACT

BACKGROUND: Our previous study showed that SUMO1 expression is closely related to progression in non-small cell lung cancer (NSCLC); however, the function of SUMO1 in NSCLC has not yet been well elucidated. METHODS: SUMO1 was enhanced or silenced in two NSCLC cell lines by using either forced SUMO1 expression or short hairpin RNA against SUMO1 lentiviral vectors, respectively. The biological functions of SUMO1 in NSCLC were investigated through colony-formation, cell proliferation, and invasion assays, and cell cycle analysis. NF-κB expression was detected in the overexpressed and silenced SUMO1 cell lines. Immunohistochemistry was used to detect an association between SUMO1 and NF-κB in the cancer and adjacent tissues of 168 patients with lung cancer. RESULTS: Overexpressed SUMO1 promoted the proliferation rate, colony formation ability, invasion, and NF-κB expression in an A549 cell line. Conversely, SUMO1 depletion inhibited the cell growth rate, colony formation ability, invasion, and NF-κB expression in a Calu-1 cell line. SUMO1 expression was significantly correlated with NF-κB expression in lung adenocarcinoma and squamous carcinoma patients (r > 0.5, P < 0.001). CONCLUSION: Our results provide evidence that SUMO1 promotes the proliferation and invasion of NSCLC cells by regulating NF-κB.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Cell Proliferation/genetics , NF-kappa B/genetics , SUMO-1 Protein/genetics , A549 Cells , Carcinoma, Non-Small-Cell Lung/pathology , Cell Cycle/genetics , Cell Movement/genetics , Female , Gene Expression Regulation, Neoplastic/genetics , Humans , Male , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , RNA, Small Interfering/genetics , SUMO-1 Protein/antagonists & inhibitors , Signal Transduction/genetics
9.
Toxicol Appl Pharmacol ; 345: 1-9, 2018 04 15.
Article in English | MEDLINE | ID: mdl-29524504

ABSTRACT

BACKGROUND AND PURPOSE: Protein modification by small ubiquitin-like modifier (SUMO) plays a critical role in the pathogenesis of heart diseases. The present study was designed to determine whether ginkgolic acid (GA) as a SUMO-1 inhibitor exerts an inhibitory effect on cardiac fibrosis induced by myocardial infarction (MI). EXPERIMENTAL APPROACH: GA was delivered by osmotic pumps in MI mice. Masson staining, electron microscopy (EM) and echocardiography were used to assess cardiac fibrosis, ultrastructure and function. Expression of SUMO-1, PML, TGF-ß1 and Pin1 was measured with Western blot or Real-time PCR. Collagen content, cell viability and myofibroblast transformation were measured in neonatal mouse cardiac fibroblasts (NMCFs). Promyelocytic leukemia (PML) protein was over-expressed by plasmid transfection. KEY RESULTS: GA improved cardiac fibrosis and dysfunction, and decreased SUMO-1 expression in MI mice. GA (>20 µM) inhibited NMCF viability in a dose-dependent manner. Nontoxic GA (10 µM) restrained angiotensin II (Ang II)-induced myofibroblast transformation and collagen production. GA also inhibited expression of TGF-ß1 mRNA and protein in vitro and in vivo. GA suppressed PML SUMOylation and PML nuclear body (PML-NB) organization, and disrupted expression and recruitment of Pin1 (a positive regulator of TGF-ß1 mRNA), whereas over-expression of PML reversed that. CONCLUSIONS AND IMPLICATIONS: Inhibition of SUMO-1 by GA alleviated MI-induced heart dysfunction and fibrosis, and the SUMOylated PML/Pin1/TGF-ß1 pathway is crucial for GA-inhibited cardiac fibrosis.


Subject(s)
Myocardial Infarction/drug therapy , SUMO-1 Protein/antagonists & inhibitors , Salicylates/therapeutic use , Animals , Animals, Newborn , Cell Survival/drug effects , Cell Survival/physiology , Dose-Response Relationship, Drug , Fibrosis/drug therapy , Fibrosis/metabolism , Fibrosis/pathology , Male , Mice , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , SUMO-1 Protein/metabolism , Salicylates/pharmacology , Stroke Volume/drug effects , Stroke Volume/physiology
10.
Cell Physiol Biochem ; 45(3): 883-898, 2018.
Article in English | MEDLINE | ID: mdl-29421780

ABSTRACT

BACKGROUND/AIMS: The myocardial sarcoplasmic reticulum calcium ATPase (SERCA2a) is a pivotal pump responsible for calcium cycling in cardiomyocytes. The present study investigated the effect of luteolin (Lut) on restoring SERCA2a protein level and stability reduced by myocardial ischemia/reperfusion (I/R) injury. We verified a hypothesis that Lut protected against myocardial I/R injury by regulating SERCA2a SUMOylation. METHODS: The hemodynamic data, myocardial infarct size of intact hearts, apoptotic analysis, mitochondrial membrane potential (ΔΨm), the level of SERCA2a SUMOylation, and the activity and expression of SERCA2a were examined in vivo and in vitro to clarify the cardioprotective effects of Lut after SUMO1 was knocked down or over-expressed. The putative SUMO conjugation sites in mouse SERCA2a were investigated as the possible regulatory mechanism of Lut. RESULTS: Initially, we found that Lut reversed the SUMOylation and stability of SERCA2a as well as the expression of SUMO1, which were reduced by I/R injury in vitro. Furthermore, Lut increased the expression and activity of SERCA2a partly through SUMO1, thus improving ΔΨm and reducing apoptotic cells in vitro and promoting the recovery of heart function and reducing infarct size in vivo. We also demonstrated that SUMO acceptor sites in mouse SERCA2a involving lysine 585, 480 and 571. Among the three acceptor sites, Lut enhanced SERCA2a stability via lysine 585. CONCLUSIONS: Our results suggest that Lut regulates SERCA2a through SUMOylation at lysine 585 to attenuate myocardial I/R injury.


Subject(s)
Luteolin/therapeutic use , Myocardial Reperfusion Injury/prevention & control , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Animals , Apoptosis/drug effects , Cell Hypoxia , Cell Line , L-Lactate Dehydrogenase/blood , Luteolin/pharmacology , Lysine/chemistry , Lysine/metabolism , Male , Membrane Potential, Mitochondrial/drug effects , Mice , Mice, Inbred C57BL , Myocardial Reperfusion Injury/pathology , Myocardium/metabolism , Myocardium/pathology , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , SUMO-1 Protein/antagonists & inhibitors , SUMO-1 Protein/genetics , SUMO-1 Protein/metabolism , Sumoylation/drug effects , Transfection , Up-Regulation/drug effects , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism
11.
Cytokine ; 102: 173-180, 2018 02.
Article in English | MEDLINE | ID: mdl-29221668

ABSTRACT

Oxidative stress-induced mitochondrial function and cell apoptosis to osteoblasts, plays a critical role in the pathophysiology of osteoporosis. However, mechanisms underlying such process remain not yet clear. We aims in this study to investigate a possible role of SMAD (the mothers against decapentaplegic homolog 4 (SMAD4) in the oxidative stress-induced apoptosis, in homo sapiens osteoblast hFOB1.19 cells. Results demonstrated that the treatment with more than 100µM H2O2 significantly downregulated the cellular viability, whereas markedly induced apoptosis in hFOB1.19 cells. The SMAD4 was markedly reduced in both mRNA and protein levels in the H2O2 -treated hFOB1.19 cells, along with the reduction of Small ubiquitin-related modifier 1 (SUMO 1) and SUMO 2/3. The immunoprecipitation assay confirmed indicated the interaction between SUMO 1 (or SUMO 2/3) and SMAD4. Moreover, the SMAD4 overexpression markedly ameliorated the H2O2-resulted viability reduction and apoptosis induction in hFOB1.19 cells. Interestingly, such amelioration was blocked by the knockdown of SUMO 2/3. Taken together, we conclued that SMAD4 inhibits the H2O2-induced apoptosis in osteoblast hFOB1.19 cells; such inhibition might depend on the SUMOylation by SUMO 2/3. It implies a promising role of SMAD4 in oxidative stress-promoted damage to osteoblasts.


Subject(s)
Apoptosis/physiology , Osteoblasts/cytology , Osteoblasts/metabolism , Smad4 Protein/metabolism , Apoptosis/drug effects , Cell Line , Gene Knockdown Techniques , Humans , Hydrogen Peroxide/toxicity , Models, Biological , Osteoblasts/drug effects , Oxidative Stress , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , SUMO-1 Protein/antagonists & inhibitors , SUMO-1 Protein/genetics , SUMO-1 Protein/metabolism , Signal Transduction , Smad4 Protein/chemistry , Smad4 Protein/genetics , Small Ubiquitin-Related Modifier Proteins/antagonists & inhibitors , Small Ubiquitin-Related Modifier Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/metabolism , Sumoylation , Ubiquitins/metabolism
12.
Sci Signal ; 10(505)2017 Nov 14.
Article in English | MEDLINE | ID: mdl-29138295

ABSTRACT

Because protein-protein interactions underpin most biological processes, developing tools that target them to understand their function or to inform the development of therapeutics is an important task. SUMOylation is the posttranslational covalent attachment of proteins in the SUMO family (SUMO-1, SUMO-2, or SUMO-3), and it regulates numerous cellular pathways. SUMOylated proteins are recognized by proteins with SUMO-interaction motifs (SIMs) that facilitate noncovalent interactions with SUMO. We describe the use of the Affimer system of peptide display for the rapid isolation of synthetic binding proteins that inhibit SUMO-dependent protein-protein interactions mediated by SIMs both in vitro and in cells. Crucially, these synthetic proteins did not prevent SUMO conjugation either in vitro or in cell-based systems, enabling the specific analysis of SUMO-mediated protein-protein interactions. Furthermore, through structural analysis and molecular modeling, we explored the molecular mechanisms that may underlie their specificity in interfering with either SUMO-1-mediated interactions or interactions mediated by either SUMO-2 or SUMO-3. Not only will these reagents enable investigation of the biological roles of SUMOylation, but the Affimer technology used to generate these synthetic binding proteins could also be exploited to design or validate reagents or therapeutics that target other protein-protein interactions.


Subject(s)
Peptide Library , Protein Interaction Domains and Motifs/drug effects , SUMO-1 Protein/metabolism , Small Molecule Libraries/pharmacology , Small Ubiquitin-Related Modifier Proteins/metabolism , Sumoylation/drug effects , Ubiquitins/metabolism , Fluorescent Antibody Technique , HEK293 Cells , Humans , Models, Molecular , Molecular Dynamics Simulation , Peptide Fragments/pharmacology , SUMO-1 Protein/antagonists & inhibitors , Small Ubiquitin-Related Modifier Proteins/antagonists & inhibitors , Ubiquitins/antagonists & inhibitors
13.
Cell Physiol Biochem ; 41(3): 987-998, 2017.
Article in English | MEDLINE | ID: mdl-28222440

ABSTRACT

BACKGROUND: It has been reported that blocking small ubiquitin-like modifier (SUMO) conjugation by silencing SUMO gene remarkably decreased tumor growth in vivo. However, few studies have examined the relationship between SUMO gene silencing and gastric cancer (GC). The study aims to explore the effects of SUMO-1 gene silencing on GC cell proliferation and apoptosis. METHODS: GC cells were cultured and divided into 5 groups: the blank group (without any transfection or treatment), the empty vector group (transfected with empty vector), the shRNA-SUMO-1-1 group (transfected with shRNA-SUMO-1-1 plasmid), the shRNA-SUMO-1-2 group (transfected with shRNA-SUMO-1-2 plasmid), and the shRNA-SUMO-1-3 group (transfected with shRNA-SUMO-1-3 plasmid). Cell Counting Kit-8 (CCK-8) assay was performed to examine cell proliferation. Annexin V/PI staining combined with flow cytometry were used to detect cell apoptosis. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were employed to measure the mRNA and protein expressions of SUMO-1, P53, Bcl-2 and c-myc, respectively. RESULTS: SUMO-1 mRNA and protein expressions were decreased after transfecting with shRNA-SUMO-1. Compared with the blank group, the shRNA-SUMO-1-1 group presented a remarkable decreased proliferation of SGC-7901 cells. Significant increase in cell apoptosis rate was observed. Bcl-2, c-myc and P53 expressions were declined after transfecting with shRNA-SUMO plasmid. CONCLUSION: Our study provided evidence that SUMO-1 gene silencing could decrease proliferation and promote apoptosis in GC cells.


Subject(s)
Apoptosis/genetics , Gastric Mucosa/metabolism , Gene Expression Regulation, Neoplastic , RNA, Messenger/antagonists & inhibitors , SUMO-1 Protein/antagonists & inhibitors , Cell Line, Tumor , Cell Proliferation , Gastric Mucosa/pathology , Humans , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , SUMO-1 Protein/genetics , SUMO-1 Protein/metabolism , Signal Transduction , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
14.
Bioorg Med Chem Lett ; 26(17): 4237-40, 2016 09 01.
Article in English | MEDLINE | ID: mdl-27491710

ABSTRACT

Several p-terphenyl compounds have been isolated from the edible Chinese mushroom Thelephora vialis. Vialinin A, a p-terphenyl compound, strongly inhibits tumor necrosis factor-α production and release. Vialinin A inhibits the enzymatic activity of ubiquitin-specific peptidase 5, one of the target molecules in RBL-2H3 cells. Here we examined the inhibitory effect of p-terphenyl compounds, including vialinin A, against sentrin/SUMO-specific protease 1 (SENP1) enzymatic activity. The half maximal inhibitory concentration values of vialinin A and thelephantin G against full-length SENP1 were 1.64±0.23µM and 2.48±0.02µM, respectively. These findings suggest that p-terphenyl compounds are potent SENP1 inhibitors.


Subject(s)
SUMO-1 Protein/metabolism , Terphenyl Compounds/metabolism , Tumor Necrosis Factor-alpha/metabolism , Agaricales/chemistry , Agaricales/metabolism , Animals , Cell Line , Humans , Kinetics , Protein Binding , Rats , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , SUMO-1 Protein/antagonists & inhibitors , Terphenyl Compounds/chemistry , Tumor Necrosis Factor-alpha/antagonists & inhibitors
15.
J Virol ; 90(13): 5939-5952, 2016 07 01.
Article in English | MEDLINE | ID: mdl-27099310

ABSTRACT

UNLABELLED: Aspects of intrinsic antiviral immunity are mediated by promyelocytic leukemia nuclear body (PML-NB) constituent proteins. During herpesvirus infection, these antiviral proteins are independently recruited to nuclear domains that contain infecting viral genomes to cooperatively promote viral genome silencing. Central to the execution of this particular antiviral response is the small ubiquitin-like modifier (SUMO) signaling pathway. However, the participating SUMOylation enzymes are not fully characterized. We identify the SUMO ligase protein inhibitor of activated STAT1 (PIAS1) as a constituent PML-NB protein. We show that PIAS1 localizes at PML-NBs in a SUMO interaction motif (SIM)-dependent manner that requires SUMOylated or SUMOylation-competent PML. Following infection with herpes simplex virus 1 (HSV-1), PIAS1 is recruited to nuclear sites associated with viral genome entry in a SIM-dependent manner, consistent with the SIM-dependent recruitment mechanisms of other well-characterized PML-NB proteins. In contrast to that of Daxx and Sp100, however, the recruitment of PIAS1 is enhanced by PML. PIAS1 promotes the stable accumulation of SUMO1 at nuclear sites associated with HSV-1 genome entry, whereas the accumulation of other evaluated PML-NB proteins occurs independently of PIAS1. We show that PIAS1 cooperatively contributes to HSV-1 restriction through mechanisms that are additive to those of PML and cooperative with those of PIAS4. The antiviral mechanisms of PIAS1 are counteracted by ICP0, the HSV-1 SUMO-targeted ubiquitin ligase, which disrupts the recruitment of PIAS1 to nuclear domains that contain infecting HSV-1 genomes through mechanisms that do not directly result in PIAS1 degradation. IMPORTANCE: Adaptive, innate, and intrinsic immunity cooperatively and efficiently restrict the propagation of viral pathogens. Intrinsic immunity mediated by constitutively expressed cellular proteins represents the first line of intracellular defense against infection. PML-NB constituent proteins mediate aspects of intrinsic immunity to restrict herpes simplex virus 1 (HSV-1) as well as other viruses. These proteins repress viral replication through mechanisms that rely on SUMO signaling. However, the participating SUMOylation enzymes are not known. We identify the SUMO ligase PIAS1 as a constituent PML-NB antiviral protein. This finding distinguishes a SUMO ligase that may mediate signaling events important in PML-NB-mediated intrinsic immunity. Moreover, this research complements the recent identification of PIAS4 as an intrinsic antiviral factor, supporting a role for PIAS proteins as both positive and negative regulators of host immunity to virus infection.


Subject(s)
Herpesvirus 1, Human/immunology , Host-Pathogen Interactions , Immunity, Innate , Promyelocytic Leukemia Protein/chemistry , Protein Inhibitors of Activated STAT/metabolism , Fibroblasts/virology , Foreskin , Gene Expression Regulation, Viral , Genome, Viral , Humans , Male , Promyelocytic Leukemia Protein/metabolism , Protein Inhibitors of Activated STAT/genetics , STAT1 Transcription Factor/chemistry , STAT1 Transcription Factor/metabolism , SUMO-1 Protein/antagonists & inhibitors , Sumoylation , Ubiquitin-Protein Ligases/metabolism , Viral Proteins/metabolism , Virus Internalization , Virus Replication
16.
Mol Reprod Dev ; 81(8): 712-24, 2014 Aug.
Article in English | MEDLINE | ID: mdl-25123474

ABSTRACT

Small ubiquitin-related modifier-1 (SUMO-1)-dependent modifications of many target proteins are involved in a range of intracellular processes. Previous studies reported the localization of SUMO-1 during oocyte meiosis, and that overexpression of Sentrin/SUMO-specific protease 2 (SENP2), a de-SUMOylation protease, altered SUMO-modified proteins, and caused defects in metaphase-II spindle organization. In this study, we detailed the consequences of SUMO-1-mediated SUMOylation by either inhibition of SUMO-1 or UBC9 with a specific antibody or their depletion by specific siRNA microinjection. Inhibition or depletion of SUMO-1 or UBC9 in germinal vesicle (GV)-stage oocytes decreased the rates of germinal vesicle breakdown and first polar body (PB1) extrusion; caused defective spindle organization and misaligned chromosomes; and led to aneuploidy in matured oocytes. Stage-specific antibody injections suggested that SUMO-1 functions before anaphase I during PB1 extrusion. Further experiments indicated that the localization of γ-tubulin was disordered after SUMO-1 inhibition, and that SUMO-1 depletion disrupted kinetochore-microtubule attachment at metaphase I. Moreover, SUMO-1 inhibition resulted in less-condensed chromosomes, altered localization of REC8 and securin, and reduced BUBR1 accumulation at the centromere. On the other hand, overexpression of SUMO-1 in GV-stage oocytes had no significant effect on oocyte maturation. In conclusion, our results implied that SUMO-1 plays crucial roles during oocyte meiotic maturation, specifically involving spindle assembly and chromosome behavior, by regulating kinetochore-microtubule attachment and the localization of γ-tubulin, BUBR1, REC8, and securin.


Subject(s)
Chromosome Segregation/physiology , Meiosis/physiology , Oocytes/physiology , SUMO-1 Protein/physiology , Spindle Apparatus/physiology , Animals , Cell Cycle Proteins/metabolism , Mice , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , RNA Interference , RNA, Small Interfering/genetics , SUMO-1 Protein/antagonists & inhibitors , SUMO-1 Protein/metabolism , Securin/metabolism , Sumoylation , Tubulin/metabolism
17.
Biochem J ; 456(3): 385-95, 2013 Dec 15.
Article in English | MEDLINE | ID: mdl-24040933

ABSTRACT

Monobodies are small recombinant proteins designed to bind with high affinity to target proteins. Monobodies have been generated to mimic the SIM [SUMO (small ubiquitin-like modifier)-interacting motif] present in many SUMO target proteins, but their properties have not been determined in cells. In the present study we characterize the properties of two SUMO1-specific monobodies (hS1MB4 and hS1MB5) in HEK (human embyronic kidney)-293 and HeLa cells and examine their ability to purify SUMO substrates from cell lines and rat brain. Both hS1MB4 and hS1MB5 compared favourably with commercially available antibodies and were highly selective for binding to SUMO1 over SUMO2/3 in pull-down assays against endogenous and overexpressed SUMO and SUMOylated proteins. Monobodies expressed in HeLa cells displayed a nuclear and cytosolic distribution that overlaps with SUMO1. Expression of the monobodies effectively inhibited protein SUMOylation by SUMO1 and, surprisingly, by SUMO2/3, but were not cytotoxic for at least 36 h. We attribute the effects on SUMO2/3 to the role of SUMO1 in chain termination and/or monobody inhibition of the SUMO-conjugating E1 enzyme complex. Taken together, these data provide the first demonstration that monobodies represent useful new tools both to isolate SUMO conjugates and to probe cell SUMOylation pathways in vivo.


Subject(s)
Gene Expression , SUMO-1 Protein/antagonists & inhibitors , SUMO-1 Protein/metabolism , Single-Chain Antibodies/biosynthesis , Sumoylation , Animals , Antibody Specificity/genetics , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cytosol/metabolism , HEK293 Cells , HeLa Cells , Humans , Rats , SUMO-1 Protein/genetics , Single-Chain Antibodies/genetics
18.
Cell Biochem Biophys ; 67(3): 1081-7, 2013.
Article in English | MEDLINE | ID: mdl-23640307

ABSTRACT

Here we investigated whether the cellular accumulation of p53 protein caused by over-expression of small ubiquitin-related modifier-1 (SUMO-1) could be used as a predictive marker for prognosis in colon cancer. We detected SUMO-1 and p53 protein levels in 46 cases of colon cancer and adjacent tissues by immunohistochemistry and found that SUMO-1 was expressed at much higher levels in colon cancer compared with that in normal colon tissue. Immunoprecipitation and Western blot analysis revealed that the tumor suppressor p53 was present predominantly in the sumoylated rather than the non-sumoylated form in the colon cancer cell lines. A small interfering RNA targeted to SUMO-1 mRNA sequences was used to observe the levels of the p53 protein. Patients who showed high dual expressions of SUMO-1 and p53 tended to experience metastasis more frequently. These results suggest that the cellular accumulation of p53 protein caused by over-expression of SUMO-1 may be involved in tumor aggressiveness. Multivariate analysis confirmed that the high dual expression of SUMO-1 and p53 was an independent factor for evaluating prognosis. SUMO-1 may be useful as a novel target for therapy in colon cancer as well as a clinical indicator for tumor aggressiveness.


Subject(s)
Colonic Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , SUMO-1 Protein/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Adult , Aged , Caco-2 Cells , Colonic Neoplasms/pathology , Female , HCT116 Cells , Humans , Immunohistochemistry , Male , Middle Aged , Neoplasm Staging , Proportional Hazards Models , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , SUMO-1 Protein/antagonists & inhibitors , SUMO-1 Protein/metabolism , Sumoylation
19.
Chem Biol ; 20(4): 604-13, 2013 Apr 18.
Article in English | MEDLINE | ID: mdl-23601649

ABSTRACT

The dynamic, posttranslational modification of proteins with a small ubiquitin-like modifier (SUMO) tag has been recognized as an important cellular regulatory mechanism relevant to a number of cancers as well as normal embryonic development. As part of a program aimed toward the identification of inhibitors of SUMO-conjugating enzymes, we developed a microfluidic electrophoretic mobility shift assay to monitor sumoylation events in real time. We disclose herein the use of this assay to identify a cell-permeable compound capable of blocking the transfer of SUMO-1 from the E2 enzyme Ubc9 to the substrate. We screened a small collection of compounds and identified an oxygenated flavonoid derivative that inhibits sumoylation in vitro. Next, we carried out an in-depth mechanistic analysis that ruled out many common false-positive mechanisms such as aggregation or alkylation. Furthermore, we report that this flavonoid inhibits a single step in the sumoylation cascade: the transfer of SUMO from the E2 enzyme (Ubc9) thioester conjugate to the substrate. In addition to having a unique mechanism of action, this inhibitor has a discrete structure-activity relationship uncharacteristic of a promiscuous inhibitor. Cell-based studies showed that the flavonoid inhibits the sumoylation of topoisomerase-I in response to camptothecin treatment in two different breast cancer cell lines, while isomeric analogs are inactive. Importantly, this compound blocks sumoylation while not affecting ubiquitylation in cells. This work identifies a point of entry for pharmacologic inhibition of the sumoylation cascade and may serve as the basis for continued study of additional pharmacophores that modulate SUMO-conjugating enzymes such as Ubc9.


Subject(s)
Electrophoretic Mobility Shift Assay , SUMO-1 Protein/metabolism , Small Molecule Libraries/chemistry , Amino Acid Sequence , Cell Line, Tumor , Humans , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , SUMO-1 Protein/antagonists & inhibitors , SUMO-1 Protein/genetics , Small Molecule Libraries/pharmacology , Structure-Activity Relationship , Sumoylation/drug effects
20.
Proc Natl Acad Sci U S A ; 108(19): 7751-6, 2011 May 10.
Article in English | MEDLINE | ID: mdl-21518904

ABSTRACT

Discriminating closely related molecules remains a major challenge in the engineering of binding proteins and inhibitors. Here we report the development of highly selective inhibitors of small ubiquitin-related modifier (SUMO) family proteins. SUMOylation is involved in the regulation of diverse cellular processes. Functional differences between two major SUMO isoforms in humans, SUMO1 and SUMO2/3, are thought to arise from distinct interactions mediated by each isoform with other proteins containing SUMO-interacting motifs (SIMs). However, the roles of such isoform-specific interactions are largely uncharacterized due in part to the difficulty in generating high-affinity, isoform-specific inhibitors of SUMO/SIM interactions. We first determined the crystal structure of a "monobody," a designed binding protein based on the fibronectin type III scaffold, bound to the yeast homolog of SUMO. This structure illustrated a mechanism by which monobodies bind to the highly conserved SIM-binding site while discriminating individual SUMO isoforms. Based on this structure, we designed a SUMO-targeted library from which we obtained monobodies that bound to the SIM-binding site of human SUMO1 with K(d) values of approximately 100 nM but bound to SUMO2 400 times more weakly. The monobodies inhibited SUMO1/SIM interactions and, unexpectedly, also inhibited SUMO1 conjugation. These high-affinity and isoform-specific inhibitors will enhance mechanistic and cellular investigations of SUMO biology.


Subject(s)
Peptides/pharmacology , Small Ubiquitin-Related Modifier Proteins/antagonists & inhibitors , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Drug Design , Humans , In Vitro Techniques , Kinetics , Models, Molecular , Molecular Sequence Data , Peptide Library , Peptides/chemistry , Peptides/metabolism , Protein Binding , Protein Engineering , Protein Interaction Domains and Motifs , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/chemistry , Protein Isoforms/metabolism , SUMO-1 Protein/antagonists & inhibitors , SUMO-1 Protein/chemistry , SUMO-1 Protein/metabolism , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/chemistry , Small Ubiquitin-Related Modifier Proteins/metabolism , Ubiquitins/antagonists & inhibitors , Ubiquitins/chemistry , Ubiquitins/metabolism
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