Sentrin-specific Protease Research Articles

Knockdown of SENP1 inhibits HIF-1α SUMOylation and suppresses oncogenic CCNE1 in Wilms tumor

Based on our initial bioinformatics finding of the upregulated expression of sentrin-specific protease 1 (SENP1) and cyclin E1 (CCNE1) in Wilms tumor, this study aimed to illustrate the molecular mechanism of SENP1 in Wilms tumor, which involved the hypoxia-inducible factor 1α (HIF-1α)/stanniocalcin-1 (STC1)/CCNE1 axis. Wilms tumor and adjacent normal tissues were clinically collected. Gain- and loss-of-function assays were performed to evaluate the effects of the regulatory axis on malignant phenotypes of Wilms tumor cells. A mouse model of Wilms tumor xenografts was further established for in vivo substantiation. Overexpression of CCNE1 and SENP1 occurred in Wilms tumor tissues and cells. Silencing SENP1 inhibited viability and enhanced cell-cycle arrest of Wilms tumor cells. SENP1 promoted STC1 expression and upregulated CCNE1 by driving the small ubiquitin-like modifier (SUMO)ylation of HIF-1α, which ultimately promoted the malignant phenotypes of Wilms tumor cells. It was further confirmed that silencing SENP1 downregulated the expression of CCNE1 and restricted tumorigenicity of Wilms tumor cells in vivo. Taken together, SENP1 elevated STC1 expression by driving the SUMOylation of HIF-1α, thereby upregulating the expression of CCNE1 and ultimately promoting the development of Wilms tumor.

Evidence of Omics, Immune Infiltration, and Pharmacogenomic for SENP1 in the Pan-Cancer Cohort.

Sentrin specific-protease 1 (SENP1) is a protein involved in deSUMOylation that is almost overexpressed in cancer. SENP1 has a determinative role in the activation of transcription programs in the innate immune responses and the development B of and C lymphocytes. We found, SENP1 possibly plays a critical role in immune infiltration and acts as an expression marker in PAAD, ESCA, and THYM. CD4+ T cells, CD8+ T cells, and macrophages were more key-related immune cells, indicating that SENP1 might be introduced as a potential target for cancer immunotherapy. We further showed that dysregulation of SENP1 is powerfully associated with decreased patient survival and clinical stage. Total SENP1 protein also increases in cancer. SENP1 is also controlled by transcription factors (TFs) CREB1, KDM5A, REST, and YY1 that regulates apoptosis, cell cycle, cell proliferation, invasion, tumorigenesis, and metastasis. These TFs were in a positive correlation with SENP1. MiR-138–5p, miR-129-1-3p, and miR-129-2-3p also inhibit tumorigenesis through targeting of SENP1. The SENP1 expression level positively correlated with the expression levels of UBN1, SP3, SAP130, NUP98, NUP153 in 32 tumor types. SENP1 and correlated and binding genes: SAP130, NUP98, and NUP153 activated cell cycle. Consistent with this finding, drug analysis was indicated SENP1 is sensitive to cell cycle, apoptosis, and RTK signaling regulators. In the end, SENP1 and its expression-correlated and functional binding genes were enriched in cell cycle, apoptosis, cellular response to DNA damage stimulus. We found that the cell cycle is the main way for tumorigenesis by SENP1. SENP1 attenuates the effect of inhibitory drugs on the cell cycle. We also introduced effective FDA-Approved drugs that can inhibit SENP1. Therefore in the treatments in which these drugs are used, SENP1 inhibition is a suitable approach. This study supplies a wide analysis of the SENP1 across The Cancer Genome Atlas (CGA) cancer types. These results suggest the potential roles of SENP1 as a biomarker for cancer. Since these drugs and the drugs that cause to resistance are applied to cancer treatment, then these two class drugs can use to inhibition of SENP1.

SENP1 participates in Irinotecan resistance in human colon cancer cells.

Colorectal cancer is one of the most prevalent cancers in the world. Chemoresistance has always been a problem encountered in its treatment. It is known that SUMOylation may regulate protein stability and decomposition, and even affect the protein translocation and posttranslational modification in cells. Sentrin-specific protease 1 (SENP1) is involved in the maturation of SUMO protein, and on the other hand, plays a role in deSUMOylation, which dissociates the target protein from SUMO and prevents further degradation of the target protein. In this study, we established an Irinotecan (CPT-11) resistant human colon cancer LoVo strain (LoVoR-CPT-11 ) to investigate the role of SENP1 in the development of drug resistance in colorectal cancer. The abundant accumulation of SENP1 and HIF-1α proteins and the increase of SUMO pathway enzymes were observed in LoVoR-CPT-11 cells while the protein markers of proliferation, angiogenesis, and glycolysis were upregulated. Knockdown of SENP1 reduced the migration ability and trigged re-sensitivity of LoVoR-CPT-11 cells to CPT-11 treatment. The analysis of SENP1 and HIF-1α gene expressions from TCGA/GTEx datasets using the GEPIA web server showed a positive correlation between SENP1 and HIF-1α in colorectal cancer patients and the high expression of these two genes might predict a poor outcome clinically. In conclusion, SENP1 might play an important role in CPT-11 resistance in colorectal cancer. Targeting SENP1 to reduce the resistant property could be considered in prospective clinical studies.

Deneddylation by SENP8 restricts hepatitis B virus propagation.

Proteins newly synthesized from messenger RNA undergo Posttranslational modifications (PTMs) such as phosphorylation, glycosylation, methylation, and ubiquitination. These PTMs have important roles in protein stability, localization, and conformation and have been reported to be involved in hepatitis B virus (HBV) propagation. Although ubiquitination plays an essential role in HBV life cycles, the involvement of ubiquitin-like proteins (UBLs) in HBV life cycles has been understudied. Through comprehensive gain- and loss-of-function screening of UBLs, we observed that neddylation, a PTM in which neural precursor cell, expressed developmentally downregulated 8 (NEDD8) is conjugated to substrate proteins, was required for efficient HBV propagation. We also found that overexpression of sentrin-specific protease 8 (SENP8), which cleaves conjugated NEDD8, suppressed HBV propagation. Further, the catalytic activity of SENP8 was required for the suppression of HBV propagation. These results indicated that the reduction of neddylation negatively regulated HBV propagation. In addition, we demonstrated that suppression of HBV propagation via SENP8 overexpression was independent of hepatitis B protein X (HBx) and HBV promoter activity. Therefore, our data suggested that neddylation plays an important role in the late stages of HBV life cycles.

Identification of novel anti-cancer agents, applying in silico method for SENP1 protease inhibition

The SENP1 (Sentrin-Specific Protease1) is essential for desumoylation. SENP1 plays an essential role in many diseases such as cardiovascular disease, diabetes and cancer via targeting GATA2, NEMO, Pin1, SMAD4 and HIF-1α for deSUMOylation. Considering that, over expression of SENP1 was reported in cancer, thus an optional inhibitor of SENP1 can restitute the balance to the skewed system of SUMO and act as an effective therapeutic agent. The purpose of this study was to select and to sort inhibitors with a stronger binding affinity with SENP1. Molecular docking of SENP1 with natural compounds including Gallic acid, Caffeic acid, Thymoquinone, Thymol, Betaine, Alkannin, Ellagic acid, Betanin, Shikonin, Betanidin and Momordin IC was performed using AutoDock 4, then docking complexes for molecular dynamics (MD) simulation with GROMACS 4.6.5 were applied. Results with RMSD, RMSF, SASA, DSSP, gyrate, H-bond, ADMET and TOPKAT measurements, binding energy and structural features were surveyed. Among those, Gallic acid has shown the most significant results including RMSD and RMSF plots with high stability, high hydrogen bonds, high binding energy and the highest intermolecular bonds with SENP1. Gallic acid demonstrated strong connections and results of toxicity better than Momordin as control. Gallic acid is a phenolic compound which affects several pharmacological and biochemical pathways and has strong antioxidant, anti-inflammatory, antimutagenic and anticancer properties. Further research can improve the appropriate use of plant products drastically. Basic, pre-clinical and clinical research on Gallic acid may provide a roadmap for its ultimate application in the field of cancer prevention. Communicated by Ramaswamy H. Sarma

SENP2 is vital for optimal insulin signaling and insulin-stimulated glycogen synthesis in human skeletal muscle cells

Sentrin-specific protease (SENP) 2 has been suggested as a possible novel drug target for the treatment of obesity and type 2 diabetes mellitus after observations of a palmitate-induced increase in SENP2 that lead to increased fatty acid oxidation and improved insulin sensitivity in skeletal muscle cells from mice. However, no precedent research has examined the role of SENP2 in human skeletal muscle cells. In the present work, we have investigated the impact of SENP2 on fatty acid and glucose metabolism as well as insulin sensitivity in human skeletal muscle using cultured primary human myotubes. Acute (4 ​h) oleic acid oxidation was reduced in SENP2-knockdown (SENP2-KD) cells compared to control cells, with no difference in uptake. After prelabeling (24 ​h) with oleic acid, total lipid content and incorporation into triacylglycerol was decreased, while incorporation into other lipids, as well as complete oxidation and β-oxidation was increased in SENP2-KD cells. Basal glucose uptake (i.e., not under insulin-stimulated conditions) was higher in SENP2-KD cells, whereas oxidation was similar to control myotubes. Further, basal glycogen synthesis was not different in SENP2-KD myotubes, but both insulin-stimulated glycogen synthesis and AktSer473 phosphorylation was completely blunted in SENP2-KD cells. In conclusion, SENP2 plays an important role in fatty acid and glucose metabolism in human myotubes. Interestingly, it also appears to have a pivotal role in regulating myotube insulin sensitivity. Future studies should examine the role of SENP2 in regulation of insulin sensitivity in other tissues and in vivo, defining the potential for SENP2 as a drug target.

CaV2.2 (N-type) voltage-gated calcium channels are activated by SUMOylation pathways

SUMOylation is an important post-translational modification process involving covalent attachment of SUMO (Small Ubiquitin-like MOdifier) protein to target proteins. Here, we investigated the potential for SUMO-1 protein to modulate the function of the CaV2.2 (N-type) voltage-gated calcium channel (VGCC), a protein vital for presynaptic neurotransmitter release. Co-expression of SUMO-1, but not the conjugation-deficient mutant SUMO-1ΔGG, increased heterologously-expressed CaV2.2 Ca2+ current density, an effect potentiated by the conjugating enzyme Ubc9. Expression of sentrin-specific protease (SENP)-1 or Ubc9 alone, had no effect on recombinant CaV2.2 channels. Co-expression of SUMO-1 and Ubc9 caused an increase in whole-cell maximal conductance (Gmax) and a hyperpolarizing shift in the midpoint of activation (V1/2). Mutation of all five CaV2.2 lysine residues to arginine within the five highest probability (>65 %) SUMOylation consensus motifs (SCMs) (construct CaV2.2-Δ5KR), produced a loss-of-function mutant. Mutagenesis of selected individual lysine residues identified K394, but not K951, as a key residue for SUMO-1-mediated increase in CaV2.2 Ca2+ current density. In synaptically-coupled superior cervical ganglion (SCG) neurons, SUMO-1 protein was distributed throughout the cell body, axons and dendrites and presumptive presynaptic terminals, whilst SUMO-1ΔGG protein was largely confined to the cell body, in particular, the nucleus. SUMO-1 expression caused increases in paired excitatory postsynaptic potential (EPSP) ratio at short (20−120 ms) inter-stimuli intervals in comparison to SUMO-1ΔGG, consistent with an increase in residual presynaptic Ca2+ current and an increase in release probability of synaptic vesicles. Together, these data provide evidence for CaV2.2 VGCCs as novel targets for SUMOylation pathways.

The Leishmania donovani SENP Protease Is Required for SUMO Processing but Not for Viability.

The protozoan parasite Leishmania donovani is part of an early eukaryotic branch and depends on post-transcriptional mechanisms for gene expression regulation. This includes post-transcriptional protein modifications, such as protein phosphorylation. The presence of genes for protein SUMOylation, i.e., the covalent attachment of small ubiquitin-like modifier (SUMO) polypeptides, in the Leishmania genomes prompted us to investigate the importance of the sentrin-specific protease (SENP) and its putative client, SUMO, for the vitality and infectivity of Leishmania donovani. While SENP null mutants are viable with reduced vitality, viable SUMO null mutant lines could not be obtained. SUMO C-terminal processing is disrupted in SENP null mutants, preventing SUMO from covalent attachment to proteins and nuclear translocation. Infectivity in vitro is not affected by the loss of SENP-dependent SUMO processing. We conclude that SENP is required for SUMO processing, but that functions of unprocessed SUMO are critical for Leishmania viability.

SUMOylation of vascular endothelial growth factor receptor 2 inhibits the proliferation, migration, and angiogenesis signaling pathway in non-small cell lung cancer.

Vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed in non-small cell lung cancer (NSCLC), and is the main target of antiangiogenesis therapy against this disease. However, there is limited evidence regarding its regulatory mechanism. Thus, elucidating the underlying mechanism of regulation of VEGFR2 is of great value to antiangiogenesis therapy. The colocalization of VEGFR2 and small ubiquitin-like modifier 1 (SUMO1) was detected through confocal microscopy. We examined the level of VEGFR2 SUMOylation in cells and rat tissues, and its effects on the angiogenesis signaling pathway (immunoprecipitation and western blotting), as well as the proliferation (Cell Counting Kit-8 assay) and migratory ability (cell scratch and Transwell assays) of NSCLC cells. Apoptosis was evaluated through Hoechst staining. VEGFR2 and SUMO1 are colocalized in the cytoplasm. VEGFR2 can be SUMOylated through combination with SUMO1 in cells and rat tissues, and the level of VEGFR2 SUMOylation in NSCLC is higher than that observed in healthy cells and tissues. Cell proliferation, migration, and the protein levels of phosphorylated-VEGFR2/phosphorylated-Akt/phosphorylated-extracellular signal-regulated kinase 1/2 (p-VEGFR2/p-Akt/p-ERK1/2) were significantly increased in NSCLC cells transfected with VEGFR2 K1270R versus those reported in cells transfected with VEGFR2 (wild-type). The levels of p-VEGFR2/p-Akt/p-ERK1/2 protein were significantly decreased in cells transfected with sentrin-specific protease 1-targeting small interfering RNA (siSENP1) versus those recorded in nontransfected controls. VEGFR2 SUMOylation may play an important role in antiangiogenesis therapy of NSCLC. The level of VEGFR2 SUMOylation may be a prognostic marker in patients with NSCLC.

Regulation of ALS-Associated SOD1 Mutant SUMOylation and Aggregation by SENP and PIAS Family Proteins.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease specific to motor neurons. Pathogenic mutations in an ALS-associated gene encoding superoxide dismutase 1 (SOD1) have been identified in familial ALS (fALS) cases. SOD1 with fALS-linked mutations is prone to form cytotoxic aggregates that cause cellular dysfunction. We previously demonstrated that the modification of SOD1 by small ubiquitin-like modifier (SUMO) 3 enhances the aggregation of fALS-linked SOD1 mutants. SUMOylation is a reversible post-translational modification targeting lysine residues. SUMO conjugation is mediated by the enzymes E1, E2, and E3, and deconjugation is catalyzed by deSUMOylation enzymes. To understand the process of SOD1 aggregation, we examined the involvement of protein inhibitor of activated STAT (PIAS) family and sentrin-specific protease (SENP) family proteins in the SUMOylation of SOD1 mutants. We found that all four types of PIAS family proteins, E3 ligase of SUMOylation, increased SUMOylation of SOD1 mutants. Among three SENP family proteins tested, deSUMOylation enzymes, SENP1, exhibited the most efficient deconjugation effect. In co-expression experiments, PIASy and SENP1 increased and decreased the number of cells exhibiting SOD1-mutant aggregation, respectively, confirming the effect of these enzymes on SOD1 aggregation. These findings suggest that regulation of SUMOylation affects the pathogenesis of ALS.

SUMO-specific protease 2 restrains the differentiation of Th17 cells

Abstract Accumulating evidence suggests that posttranslational modifications (PTMs) play a critical role in the regulation of cellular functions and disease progression. Several reports indicated that SUMOylation, a type of PTMs, contributes to diseases through modulation of cell signaling and functions. The process of SUMOylation is dynamic and can be reversibly removed by the sentrin-specific protease (SENP) family. Smad4 has been shown to suppress the differentiation of pathogenic Th17 cells and can be deSUMOylated by SENP2. Here, we would like to study whether SUMOylation may affect T cell development and differentiation, and T cell-mediated diseases. To study the roles of Senp2 in T cell lineage, we generated Senp2f/f×Lck-cre (CKO) mice. We found that there were more Th1 and Th17 cells in CKO mice in the steady state, as compared with those in littermate controls (wild type, WT) mice. Besides, Senp2-deficiency in T cells also promotes the progression of dextran sodium sulfate (DSS)-induced colitis in mice. The levels of IFNg and IL-17 produced by in vitro differentiated Th1 and Th17 cells, respectively, were higher when Senp2 is deleted. The enzymatic activity of Senp2 is required for the inhibition of Th17 differentiation; as compared with the effects of lentiviral vector expressing wild type Senp2, transduction of differentiating CKO Th17 cells with a lentiviral vector expressing a catalytic domain mutant Senp2 failed to suppress the frequency of Th17 cells. We are examining if SUMOylation affects the function of Smad4 in Th17 cells. Results from this study may provide insights into the new therapeutic strategy for the control of Th17-mediated diseases.

Increased hypoxic proliferative response and gene expression in erythroid progenitor cells of Andean highlanders with chronic mountain sickness.

Excessive erythrocytosis (EE) is the main sign of chronic mountain sickness (CMS), a maladaptive clinical syndrome prevalent in Andean and other high-altitude populations worldwide. The pathophysiological mechanism of EE is still controversial, as physiological variability of systemic respiratory, cardiovascular, and hormonal responses to chronic hypoxemia complicates the identification of underlying causes. Induced pluripotent stem cells derived from CMS highlanders showed increased expression of genes relevant to the regulation of erythropoiesis, angiogenesis, cardiovascular, and steroid-hormone function that appear to explain the exaggerated erythropoietic response. However, the cellular response to hypoxia in native CMS cells is yet unknown. This study had three related aims: to determine the hypoxic proliferation of native erythroid progenitor burst-forming unit-erythroid (BFU-E) cells derived from CMS and non-CMS peripheral blood mononuclear cells; to examine their sentrin-specific protease 1 (SENP1), GATA-binding factor 1 (GATA1), erythropoietin (EPO), and EPO receptor (EPOR) expression; and to investigate the functional upstream role of SENP1 in native progenitor differentiation into erythroid precursors. Native CMS BFU-E colonies showed increased proliferation under hypoxic conditions compared with non-CMS cells, together with an upregulated expression of SENP1, GATA1, EPOR; and no difference in EPO expression. Knock-down of the SENP1 gene abolished the augmented proliferative response. Thus, we demonstrate that native CMS progenitor cells produce a larger proportion of erythroid precursors under hypoxia and that SENP1 is essential for proliferation. Our findings suggest a significant intrinsic component for developing EE in CMS highlanders at the cellular and gene expression level that could be further enhanced by systemic factors such as alterations in respiratory control, or differential hormonal patterns.

Genome-Wide Association Analysis for Resistance to Infectious Pancreatic Necrosis Virus Identifies Candidate Genes Involved in Viral Replication and Immune Response in Rainbow Trout (Oncorhynchus mykiss).

Infectious pancreatic necrosis (IPN) is a viral disease with considerable negative impact on the rainbow trout (Oncorhynchus mykiss) aquaculture industry. The aim of the present work was to detect genomic regions that explain resistance to infectious pancreatic necrosis virus (IPNV) in rainbow trout. A total of 2,278 fish from 58 full-sib families were challenged with IPNV and 768 individuals were genotyped (488 resistant and 280 susceptible), using a 57K SNP panel Axiom, Affymetrix. A genome-wide association study (GWAS) was performed using the phenotypes time to death (TD) and binary survival (BS), along with the genotypes of the challenged fish using a Bayesian model (Bayes C). Heritabilities for resistance to IPNV estimated using genomic information, were 0.53 and 0.82 for TD and BS, respectively. The Bayesian GWAS detected a SNP located on chromosome 5 explaining 19% of the genetic variance for TD. The proximity of Sentrin-specific protease 5 (SENP5) to this SNP makes it a candidate gene for resistance against IPNV. In case of BS, a SNP located on chromosome 23 was detected explaining 9% of the genetic variance. However, the moderate-low proportion of variance explained by the detected marker leads to the conclusion that the incorporation of all genomic information, through genomic selection, would be the most appropriate approach to accelerate genetic progress for the improvement of resistance against IPNV in rainbow trout.

Salivary proteomics of canine oral tumors using MALDI-TOF mass spectrometry and LC-tandem mass spectrometry.

Canine oral tumors are relatively common neoplasms in dogs. For disease monitoring and early diagnosis, salivary biomarkers are appropriate because saliva collection is non-invasive and requires no professional skills. In the era of omics, matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF MS) coupled with liquid chromatography-tandem MS (LC-MS/MS) are suitable to identify potential disease-associated peptides and proteins. The present study aimed to use MALDI-TOF MS and LC-MS/MS to search for particular peptide mass fingerprints (PMFs) and conceivable biomarkers in saliva of dogs with early- and late-stage oral melanoma (EOM and LOM, respectively), oral squamous cell carcinoma (OSCC), benign oral tumors (BN), and periodontitis and healthy controls (CP). Pooled saliva samples in each group were used to be representative of population change. Unique PMFs were obtained and specific peptide fragments were sequenced by LC-MS/MS and BLAST-searched with mammalian protein databases. Seven peptide fragments appeared in the tumor groups (EOM, LOM, OSCC and BN) at 1096, 1208, 1322, 1794, 1864, 2354 and 2483 Da, two peptide fragments appeared in the LOM and OSCC groups at 2450 and 3492 Da, and in the CP controls at 2544 and 3026 Da. Also, protein–chemotherapy drug interaction networks were exhibited. Using western blot analysis, the expression of sentrin-specific protease 7 (SENP7), a peptide fragment at 1096 Da, in OSCC was significantly increased, as was the expression of TLR4, a peptide fragment at 3492 Da, in LOM and OSCC, compared with the CP group. The expression of nuclear factor kappa B (NF-κB), a TLR4 partner, was notably increased in OSCC compared with CP, BN and EOM. The expression was also enhanced in LOM compared with EOM. Expressed protein sequences from western blots were verified by LC-MS/MS. Western blots were then performed with individual samples in each group. The results showed the elevated expression of TLR4 in LOM and OSCC, compared with that in CP and BN, the increased expression of NF-κB in LOM and OSCC, compared with CP and in LOM compared with BN, and the enhanced expression of SENP7 in LOM and OSCC, compared with that in CP and BN. In conclusion, discrete clusters of EOM, LOM, OSCC, BN and CP groups and potential protein candidates associated with the diseases were demonstrated by salivary proteomics. Western blot analysis verified SENP7, TLR4 and NF-κB as potential salivary biomarkers of canine oral tumors.

Ribosomal Protein S9 Expression in Multiple Myeloma and Its Effect on Cell Biological Function

Objective To identify the expression of ribosomal protein S9(RPS9)in multiple myeloma(MM)and explore its effect on the biological characteristics of myeloma cells and the corresponding mechanisms. Methods Bone marrow mononuclear cells were harvested in 10 healthy volunteers(CON group)and bone marrow CD138 +cells from 30 MM patients(CD138+group).Quantitative polymerase chain reaction(qPCR)was performed to detect RPS9 expression at mRNA level.In three cases from CON group and 11 cases from CD138+group,Western blot was performed to detect RPS9 at protein level.GSE19784 dataset was employed to detect the relationships of RPS9 expression with the overall survival rate,nuclear factor-κB(NF-κB),small ubiquitin-like modifier(SUMO),and ubiquitin pathway.After the RPS9 knock-down vector was constructed,flow cytometry was performed to detect the infection efficiency and qPCR and Western blot to detect the knock-down efficiency.RPMI8226 was divided into CON group and RPS9-short hairpin RNA(shRNA)group,in which annexin V allophycocyanin/propidium iodide(PI)double staining was performed to detect the change of apoptosis,CCK8 to detect the proliferation change,and PI staining to detect cell cycle change.After sentrin-specific protease 1(SENP1)overexpression vector was constructed,Western blot was performed to detect the phosphorylation of P65 and inhibitory subunit-κBα(IκBα)from NF-κB pathway in CON,RPS9-shRNA,and RPS9-shRNA-SENP1 cells;in addition,annexin V/PI double staining was also performed to detect the apoptosis in these three cells. Results The relative expression of RPS9 in CON group and CD138+group was(1.00±0.12)and(5.45±0.71),respectively(t=4.291,P=0.0036).Western blot showed RPS9 expression was high in most myeloma CD138+cells.The high expression of RPS9 was associated with both extramedullary invasion and overall survival in GSE19784 dataset.After RPMI8226 was infected with CON or RPS9-shRNA lentivirus for 48 hours,flow cytometry confirmed that the infection efficiencies were above 90% in both groups.qPCR and Western blot confirmed that RPS9 expression was inhibited at both mRNA and protein levels.After RPMI8226 CON and RPS9-shRNA infected with virus for 48 hours,the proportion of annexin V-positive cells in CON and RPS9-shRNA cells was(3.47±0.37)% and(18.60±64.00)%(t=9.015,P=0.0008).The proliferation index significantly differed between CON group and RPS9-shRNA group at 72 hours(t=6.846,P=0.0024).When CON and RPS9-shRNA were infected with virus for 48 hours,the proportion of G2 phase cells was(29.28±3.42)% and(10.43±1.43)%,respectively(t=9.329,P=0.0007).The RPS9 expression was positively correlated with SENP1 in GSE19784 dataset and negatively correlated with IκBα coding gene NFKBIA.Western blot further confirmed that RPS9 knockdown inhibited the expression of SENP1,inhibited the phosphorylation of NF-κB subunit P65 and inhibitor IκBα,and promoted the expression of IκBα.Overexpression of SENP1 not only impeded this effect but also reduced RPS9-induced apoptosis. Conclusions RPS9 is highly expressed in MM CD138+cells and is associated with overall survival and extramedullary infiltration.Inhibition of RPS9 can promote apoptosis,cell cycle arrest,and proliferation of myeloma cells.RPS9 can affect the activation of NF-κB pathway and cell apoptosis through SENP1,suggesting that SENP1 may be a key factor in the biological effect of RPS9.

Noncovalent structure of SENP1 in complex with SUMO2.

SUMOylation is a post-translational modification in which a small ubiquitin-like molecule (SUMO) is appended to substrate proteins and is known to influence myriads of biological processes. A delicate interplay between several families of SUMOylation proteins and their substrates ensures the proper level of SUMOylation required for normal cell function. Among the SUMO proteins, SUMO2 is known to form mono-SUMOylated proteins and engage in poly-SUMO chain formation, while sentrin-specific protease 1 (SENP1) is a key enzyme in regulating both events. Determination of the SENP1-SUMO2 interaction is therefore necessary to better understand SUMOylation. In this regard, the current paper reports the noncovalent structure of SENP1 in complex with SUMO2, which was refined to a resolution of 2.62 Å with R and Rfree values of 22.92% and 27.66%, respectively. The structure shows that SENP1-SUMO2 complex formation is driven largely by polar interactions and limited hydrophobic contacts. The essential C-terminal motif (QQTGG) of SUMO2 is stabilized by a number of specific bonding interactions that enable it to protrude into the catalytic triad of SENP1 and provide the arrangement necessary for maturation of SUMO and deSUMOylation activity. Overall, the structure shows a number of structural details that pinpoint the basis of SENP1-SUMO2 complex formation.

Overexpressions of sentrin-specific protease-1 is associated with the invasion and metastasis of human hepatocellular carcinoma

Objective To investigate the expressions of sentrin specific protease-1 (SENP-1) in hepatocellular carcinoma (HCC), and explore its correlations with tumor metastasis. Methods The expression of SENP-1 and hypoxia inducible factor-1α (HIF-1α) were assessed by polymerase chain reaction (PCR)and immunohistochemical staining in human hepatocellular carcinoma, edge tissues and non-cancer tissues, microvascular density (MVD)was determined. The expressions of SENP-1 in HCC cell lines MHCC97H, MHCC97L and HepG2 were assessed by reverse transcriptase-PCR (RT-PCR), quantitative real-time quantitative PCR (Real-time PCR) and Western blotting. The plasmid SENP1-small interfering RNA (siRNA) conducted was used to transfected into MHCC97H cells. Western blotting analysis was identified for the effect of transfection. The expression of cysteinyl aspartate specific proteinase-8 (Caspase-8), B-cell lymphoma-2 (bcl-2), HIF-1α and vascular endothelial growth factor (VEGF) protein in hepatoma cells before and after transfection was detected by Western blotting. The invasion and metastasis of hepatoma cells were detected by wound assay and Transwell invasion assay. Results Compared with the hepatocellular carcinoma tissues and non-cancer tissues, the expression of SENP-1 and HIF-1α in tumor-surrounding tissues were much higher. Compared with immortalized liver cell line, the HCC cell lines showed higher expression of SENP1 mRNA and protein, the expressions of SENP-1 was positive correlation with the metastasis ability of HCC cell lines. After transfection of SENP1-siRNA plasmid into MHCC97H cells, the expression of apoptosis promoting protein Caspase-8 was increased, and the expression of apoptotic protein bcl-2 was inhibited. Scratch healing assay revealed that the scuffing distance of hepatoma cells after transfection for 24 h was significantly increased compared with non-treatment group. Transwel1 assay showed that the number of hepatoma cells that migrated through the matrigel after transfection for 24 h was significantly decreased compared with non-treatment group (P=0.000). Conclusion The high expressions of SENP-1 in HCC might play an important role in tumor metastasis. The mechanism may be related to the over expression of SENP-1, which promotes liver cancer cells to adapt to hypoxic microenvironment and inhibit cell apoptosis. Key words: Sentrin specific peptidase 1; Hepatocellular carcinoma; Metastasis

The role of sentrin-specific protease 2 substrate recognition in TGF-\u03b2-induced tumorigenesis

Smad4, a common-mediator of Smads, plays a central role in forming complexes with receptor-phosphorylated Smads, and then transduces transforming growth factor (TGF)-β signals into the nuclei. Although many cellular factors are involved in TGF-β induced epithelial-to-mesenchymal transition (EMT) and cell migration, very little is known with the mechanism of Smad4 regulation on pro-oncogenes response by TGF-β. Herein, we demonstrate the interaction of Sentrin-specific protease 2 (SENP2) with Smad4 through SENP2 residue 363~400. The same segment is also important for desumoylation of Smad4, and able to relieve sumoylation-mediated TGF-β repression. The SENP2363~400 segment is critical for TGF-β-induced cell migration, which is correlated with SENP2363~400 deletion mutant failed to increase matrix metalloproteinase (MMP)-9 and EMT marker gene expression. Moreover, our results suggest that the interaction and desumoylation between SENP2 and Smad4 promote cell migration in triple-negative breast cancer cells. Altogether, our data show how SENP2 regulates its substrate for desumoylation, and also the role of SENP2 in TGF-β induced cancer cell migration.

SUMO-specific proteases and isopeptidases of the SENP family at a glance.

The ubiquitin-related SUMO system controls many cellular signaling networks. In mammalian cells, three SUMO forms (SUMO1, SUMO2 and SUMO3) act as covalent modifiers of up to thousands of cellular proteins. SUMO conjugation affects cell function mainly by regulating the plasticity of protein networks. Importantly, the modification is reversible and highly dynamic. Cysteine proteases of the sentrin-specific protease (SENP) family reverse SUMO conjugation in mammalian cells. In this Cell Science at a Glance article and the accompanying poster, we will summarize how the six members of the mammalian SENP family orchestrate multifaceted deconjugation events to coordinate cell processes, such as gene expression, the DNA damage response and inflammation.

MicroRNA-186 Suppresses Cell Proliferation and Metastasis Through Targeting Sentrin-Specific Protease 1 in Renal Cell Carcinoma.

Recent evidence suggests that dysregulation of microRNAs is associated with the development of multiple malignancies. miR-186 has been reported as a critical cancer regulator in several types of cancers. However, its functional significance and molecular mechanism underlying renal cell carcinoma (RCC) remain unknown. In this study, our results showed that miR-186 expression was dramatically downregulated in RCC tissues and cell lines compared to that in adjacent normal tissues and cell lines. Overexpression of miR-186 significantly inhibited cell growth, colony formation, and cell invasion; caused cell cycle arrest at the G0/G1 phase; and induced cell apoptosis as detected by MTT, colony formation, Transwell assay, and flow cytometry assays in RCC cells. In addition, inhibition of miR-186 expression promoted RCC cell proliferation, invasion, and cell cycle progression and reduced apoptosis. Bioinformatics analysis and luciferase reporter assay confirmed that the 3′-UTR of sentrin-specific protease 1 (SENP1) was a direct target of miR-186. A remarkably reverse correlation was observed between miR-186 and SENP1 mRNA in RCC tissues. Furthermore, immunohistochemical staining revealed that SENP1 was positively expressed in RCC specimens. Restoration of SENP1 expression could partially abrogate the inhibitory effect of miR-186 overexpression on RCC cell proliferation through activating NF-κB signaling and its downstream proteins. These data demonstrated that miR-186 acted as a novel tumor suppressor and potential therapeutic biomarker in the progression of RCC by directly targeting SENP1.