Regulation mechanism of post-translational modification of farnesoid X receptor / 中国临床药理学与治疗学

Farnesoid X receptor (FXR) is a nuclear receptor activated by bile acid that is involved in regulating gene expression related to bile acid, fat, glucose, and amino acid metabolism. The activity of FXR is regulated by a variety of post-translational modifications. Common post-translational modifications of FXR include O-GlcNAcylation, phosphorylation, acetylation, sumoylation, methylation, etc. These post-translational modifications may affect FXR binding of DNA and ligand, heterodimerization, and subcellular localization, and may specifically regulate downstream gene transcription and expression. Different post-translational modifications can lead to changes in FXR stability and biological function, which are closely related to the occurrence of diseases. This paper aims to review the post-translational modification of FXR in the past five years and the mechanisms involved in disease regulation, to explore the effects of post-translational modification on the physiological function of FXR and to provide a theoretical basis for mechanism research targeting FXR.

Sortilin-induced lipid accumulation and atherogenesis are suppressed by HNF1b SUMOylation promoted by flavone of Polygonatum odoratum / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

This study aims to investigate the impact of hepatocyte nuclear factor 1β (HNF1b) on macrophage sortilin-mediated lipid metabolism and aortic atherosclerosis and explore the role of the flavone of Polygonatum odoratum (PAOA-flavone)-promoted small ubiquitin-related modifier (SUMO) modification in the atheroprotective efficacy of HNF1b. HNF1b was predicted to be a transcriptional regulator of sortilin expression via bioinformatics, dual-luciferase reporter gene assay, and chromatin immunoprecipitation. HNF1b overexpression decreased sortilin expression and cellular lipid contents in THP-1 macrophages, leading to a depression in atherosclerotic plaque formation in low-density lipoprotein (LDL) receptor-deficient (LDLR-/-) mice. Multiple SUMO1-modified sites were identified on the HNF1b protein and co-immunoprecipitation confirmed its SUMO1 modification. The SUMOylation of HNF1b protein enhanced the HNF1b-inhibited effect on sortilin expression and reduced lipid contents in macrophages. PAOA-flavone treatment promoted SUMO-activating enzyme subunit 1 (SAE1) expression and SAE1-catalyzed SUMOylation of the HNF1b protein, which prevented sortilin-mediated lipid accumulation in macrophages and the formation of atherosclerotic plaques in apolipoprotein E-deficient (ApoE-/-) mice. Interference with SAE1 abrogated the improvement in lipid metabolism in macrophage cells and atheroprotective efficacy in vivo upon PAOA-flavone administration. In summary, HNF1b transcriptionally suppressed sortilin expression and macrophage lipid accumulation to inhibit aortic lipid deposition and the development of atherosclerosis. This anti-atherosclerotic effect was enhanced by PAOA-flavone-facilitated, SAE1-catalyzed SUMOylation of the HNF1b protein.

The effect of SUMOylated peroxisome proliferator-activated receptor γ in the regulation of diabetes mellitus accelerated atherosclerosis / 中国医师进修杂志

Objective:To explore the role of SUMOylaiton of peroxisome proliferator-activated receptor γ (PPARγ) in diabetes mellitus prompted inflammation and atherosclerosis in vascular and endothelial cells.Methods:From September 2014 to January 2017, 32 Sprague-Dawley rats in 14 weeks-old were divided into sham operated group, artery injured without diabetes group, artery injured with diabetes group and ubiquitin-conjugating enzyme 9 (UBC9) transfection group (Group D) by random digits table method with 8 rats each. Model of type 1 diabetes mellitus (T1DM) and rat carotid artery balloon injury was made in the assigned group. One rat was excluded because of model failure in each group. Systolic and diastolic common carotid artery diameter and intimal thickness of injured and healthy common carotid artery were evaluated by vascular ultrasound, and the standardized common carotid artery diastolic diameter (sCADD) was calculated. Histological tests and immunohistochemical staining were performed to evaluate intimal hyperplasia, and the ratio of intimal area to media area was calculated when the media area was equal. Human umbilical vein endothelial cells (HUVEC) were cultured 24 h in high glucose medium with different duration and concentration, and the expression levels of interleukin (IL)-8 and IL-1β mRNA were determined by real time reverse transcription polymerase chain reaction (RT-PCR), the expression level of UBC9 was determined by Western blot method, SUMOylation assay kit was used to evaluate SUMOylation of PPARγ. HUVEC was cultured in vitro and PPAR was stimulated by high glucose at different concentrations and different times PPARγ SUMOylation level. UBC9 was overexpressed by lentivirus in vivo and in vitro, and the PPARγ SUMOylation level was detected.Results:The intimal thickness, intimal area and ratio of intimal area to media area 8 weeks after carotid artery injuring in sham operated group, artery injured without diabetes group and artery injured with diabetes group were increased respectively: (0.026 ± 0.018), (0.084 ± 0.007) and (0.264 ± 0.022) mm; (0.18 ± 0.09) × 10 6, (0.32 ± 0.06) × 10 6 and (1.64 ± 0.22)×10 6 μm 2; 0.345 ± 0.073, 0.570 ± 0.080 and 2.710 ± 0.220, the sCADD was decreased respectively: 0.903 ± 0.084, 0.800 ± 0.071 and 0.330 ± 0.036, and there were statistical differences ( F = 10.40, 9.40, 8.20 and 8.60; P<0.05). After HUVEC was cultured in high glucose for 24 h, the IL-8 mRNA at sugar concentrations of 10, 20 and 40 mmol/L was 1.00 ± 0.11, 3.57 ± 0.22 and 4.07 ± 0.40, the IL-1β mRNA was 1.00 ± 0.07, 3.32 ± 0.29 and 5.13 ± 0.19, and there were statistical differences ( F = 73.05 and 205.80, P<0.05). The level of PPARγ SUMOylation and UBC9 in artery injured with diabetes group were significantly lower than those in artery injured without diabetes group (0.46 ± 0.25 vs. 1.00 ± 0.21 and 0.45 ± 0.02 vs. 1.00 ± 0.07), and there were statistical differences ( P<0.05); there was no statistical difference in PPARγ between 2 groups (0.94 ± 0.07 vs. 1.00 ± 0.04, P>0.05). The UBC9 and PPARγ SUMOylation at sugar concentrations of 0, 10, 20 and 40 mmol/L were decreased respectively (0.99 ± 0.05, 0.80 ± 0.06 and 0.62 ± 0.05; 1.00 ± 0.05, 0.57 ± 0.13 and 0.55 ± 0.08), and there were statistical differences ( F = 21.02 and 14.31, P<0.05); there was no statistical difference in PPARγ (1.00 ± 0.03, 0.90 ± 0.04 and 0.91 ± 0.05; F = 3.11, P>0.05). In HUVEC cultured in high glucose medium (20 mmol/L) for 6, 12, 24 and 48 h, the UBC9 and PPARγ SUMOylation were downregulated progressively (1.00 ± 0.09, 0.75 ± 0.05, 0.70 ± 0.08, 0.38 ± 0.04 and 0.35 ± 0.03; 1.00 ± 0.03, 0.86 ± 0.01, 0.59 ± 0.01, 0.51 ± 0.11 and 0.35 ± 0.08), and there were statistical differences ( F = 36.06 and 33.13, P<0.05); but there was no statistical difference in PPARγ (1.00 ± 0.03, 1.14 ± 0.02, 1.18 ± 0.17, 0.98 ± 0.01 and 1.04 ± 0.05; F = 1.90, P>0.05). After overexpression of UBC9 in rats with diabetes, histological analysis showed that UBC9 in artery injured without diabetes group, artery injured with diabetes group and UBC9 transfection group was 1.53 ± 0.18, 1.00 ± 0.22 and 3.62 ± 0.35, there was statistical difference ( F = 5.64, P<0.05). Ultrasonic test results show that in artery injured without diabetes group, artery injured with diabetes group and UBC9 transfection group intimal thickness was increased respectively: (0.077 ± 0.015), (0.216 ± 0.007) and (0.125 ± 0.014) mm, and there was statistical difference ( F = 27.18, P<0.05). Histological analysis showed that intimal area in artery injured without diabetes group, artery injured with diabetes group and UBC9 transfection group was (0.335 ± 0.066) ×10 6, (1.053 ± 0.103) ×10 6 and (0.544 ± 0.040) ×10 6 μm 2, the ratio of intimal area to media area was 0.63 ± 0.063, 2.03 ± 0.052 and 0.93 ± 0.100, there were statistical differences ( F = 13.58 and 53.96, P<0.05). Conclusions:Diabetes mellitus could inhibit the PPARγ SUMOylaiton and prompt inflammation and atherosclerosis in vascular and endothelial cells. Upregulation of PPARγ SUMOylaiton though UBC9 overexpressioncould play a protecting role in diabetes mellitus prompted atherosclerosis.

Emerging role of protein modification in inflammatory bowel disease / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The onset of inflammatory bowel disease (IBD) involves many factors, including environmental parameters, microorganisms, and the immune system. Although research on IBD continues to expand, the specific pathogenesis mechanism is still unclear. Protein modification refers to chemical modification after protein biosynthesis, also known as post-translational modification (PTM), which causes changes in the properties and functions of proteins. Since proteins can be modified in different ways, such as acetylation, methylation, and phosphorylation, the functions of proteins in different modified states will also be different. Transitions between different states of protein or changes in modification sites can regulate protein properties and functions. Such modifications like neddylation, sumoylation, glycosylation, and acetylation can activate or inhibit various signaling pathways (e.g., nuclear factor-‍κB (NF-‍κB), extracellular signal-regulated kinase (ERK), and protein kinase B (AKT)) by changing the intestinal flora, regulating immune cells, modulating the release of cytokines such as interleukin-1β (IL-‍‍1β), tumor necrosis factor-α (TNF‍-‍α), and interferon-‍γ (IFN-‍γ), and ultimately leading to the maintenance of the stability of the intestinal epithelial barrier. In this review, we focus on the current understanding of PTM and describe its regulatory role in the pathogenesis of IBD.

Research progress on targeted SUMOylation inhibitors and their antitumor activity / 药学学报

SUMOylation is an important post-translational modification of proteins. Similar to ubiquitylation, SUMOylation is the process that the small ubiquitin-like modifier (SUMO) proteins are specifically and covalently binding to lysine residues of substrate proteins. Through SUMOylation, the physiological functions and pathological processes of cells are well controlled and balanced, and its abnormal activation has been reported in various tumors. Therefore, SUMOylation has been a potential target for anti-tumor drug development. In this review, we summarize recent advances on development of inhibitors targeting SUMOylation pathway and their antitumor properties.

KIF17 Modulates Epileptic Seizures and Membrane Expression of the NMDA Receptor Subunit NR2B / 神经科学通报·英文版

Epilepsy is a common and severe brain disease affecting >65 million people worldwide. Recent studies have shown that kinesin superfamily motor protein 17 (KIF17) is expressed in neurons and is involved in regulating the dendrite-targeted transport of N-methyl-D-aspartate receptor subtype 2B (NR2B). However, the effect of KIF17 on epileptic seizures remains to be explored. We found that KIF17 was mainly expressed in neurons and that its expression was increased in epileptic brain tissue. In the kainic acid (KA)-induced epilepsy mouse model, KIF17 overexpression increased the severity of epileptic activity, whereas KIF17 knockdown had the opposite effect. In electrophysiological tests, KIF17 regulated excitatory synaptic transmission, potentially due to KIF17-mediated NR2B membrane expression. In addition, this report provides the first demonstration that KIF17 is modified by SUMOylation (SUMO, small ubiquitin-like modifier), which plays a vital role in the stabilization and maintenance of KIF17 in epilepsy.

Exercise and Alzheimer's Disease: A Review of Research Based on Post￣translational Modification of Tau Protein / 中国生物化学与分子生物学报

Alzheimer' s disease (AD) is an age-related neurodegenerative disease which seriously damages the physical and mental health of the elderly and causes huge economic pressure to the society. However, the pathogenesis of AD is not completely elucidated. There is still no effective drug to cure AD in clinical practice. Tau protein is a soluble and non-aggregating microtubule-related protein, which can stabilize microtubule structure. The structure and function of Tau protein are abnormal in AD' s brain while under pathological conditions, and the abnormal Tau protein aggregates to insoluble neurofibrillary tangles which damages microtubules and leads to cognitive dysfunction. These changes of Tau protein are regulated by a variety of post-translational modifications, which directly change the properties and functions of proteins by attaching specific chemical moieties to Tau protein's C-terminus or N-terminus. It's confirmed that a variety of Tau post-translational modifications, like phosphorylation, glycosylation, acetylation and sumoylation is abnormal in AD's brain, which is closely related to Tau degradation and the accumulation of toxic substances. In this review, we summarized the latest progress supporting the role of exercise regulated Tau post-translational modification in the prevention and treatment of Alzheimer's disease. Firstly, exercise inhibits tau protein hyperphosphorylation by suppressing the activity of GSK-3β and MAPKs and possibly by up-regulating the activity of PP2A. Secondly, exercise increases tau protein O-GlcNAcylation by up-regulating the expression of GLUT1 and GLUT3, also possibly by regulating the balance of activity of OGT and OGA. Thirdly, exercise decreases tau protein acetylation possibly by inhibiting p300 and activating SIRT1; exercise regulates the acetylation of Tau KXGS possibly by inhibiting HDAC6. Lastly, exercise inhibits abnormal Tau sumoylation possibly by regulating the co-location sites of phosphorylation and sumoylation.

Identification of sumoylated targets in proliferating mouse spermatogonia and human testicular seminomas / 亚洲男科学杂志(英文版)

Spermatogenesis is regulated by a complex network of posttranslation modifications. Sumoylation (a modification by small ubiquitin-like modifiers, or SUMO proteins) was identified as an important cellular event in different cell types. SUMO proteins are highly expressed in the testis, and their role during spermatogenesis has begun to be elucidated. Given the important role of sumoylation in the regulation of mitosis and cancer progression in other tissues, the aim of the current study was to identify the targets of SUMO in proliferating mouse spermatogonia and human seminoma tissues and to initially examine the level of sumoylation in relation to the proliferative activity of the tissues. Using freshly purified spermatogonia and C18-4 spermatogonia cell line, mass spectrometry analysis identified several SUMO targets implicated into the proliferation of spermatogonia (such as heat shock protein 60 [HSP60] and prohibitin). Tissue array and western blot approaches showed that SUMO expression is a prominent feature of human seminomas and that the proliferative activity of the tumor tissues was positively correlated with the level of SUMO expression. Downregulation of sumoylation with si-RNA was not sufficient to significantly affect the proliferation of C18-4 spermatogonia; however, SUMO overexpression increased the proliferation rate of the cells. These data suggest that cells are more sensitive to an elevated level of SUMO, and that this situation may lead to an upregulated cellular proliferation and, possibly, cancer. Mass spectrometry analysis identified around a hundred SUMO targets in seminoma samples. Notably, many of the identified proteins (such as proliferating cell nuclear antigen [PCNA], DNA topoisomerase 2-alpha [Top2A], prohibitin, 14-3-3 protein, and others) were implicated in oncogenic transformation and cancer progression.

Research progress of SUMOylation modification in DNA double-strand break repair / 国际生物医学工程杂志

The small ubiquitin-like modified protein (SUMO) is a protein structurally similar to ubiquitin which is involved in post-translational modification of proteins. SUMOylation refers to the process that SUMO molecule covalently binding to the specific lysine site of target proteins through maturation, activation, binding and ligation by ubiquitin-like specific protease 1 (Ulp1), E1 activating enzyme, E2 binding enzyme, and E3 ligase. SUMOylation alters the activity of target proteins, which is involved in the regulation of various cellular functions such as transcriptional regulation, regulation of embryonic development, cellular stress, maintenance of chromatin structure and genomic stability. In recent years, it has been found that SUMOylation modification is also widely involved in DNA damage repair, especially DNA double-strand breaks (DSBs), which are the most serious types of DNA damage. SUMOylation is involved in almost all processes of DSBs repair, so its role in DNA damage repair has become a research hotspot. In this paper, the research progress of the regulation of SUMOylation in DSBs repair was reviewed.

Inhibition effect of SUMOylation of peroxisome proliferator activated receptor γ1 on macrophage M2 polarization / 上海交通大学学报（医学版）

Objective • To investigate the role and the regulation mechanism of SUMOylation of peroxisome proliferator activated receptor γ1 (PPARγ1) in macrophage M2 polarization induced by interleukin-4 (IL-4). Methods • To investigate the SUMOylation of PPARγ1 and identify its SUMOylated site, immunoprecipitation (IP) with anti-FLAG/HA antibody and Western blotting were used after plasmids FLAG-PPARγ1-WT/mutant and HA-SUMO1 being co-transfected into HEK293T cells. To determine SENP1 can de-SUMOylate PPARγ1, IP was used when HEK293T cells were co-transfected by FLAG-PPARγ1-WT, HA-SUMO1 and RGS-SENP1-WT, or SENP1 mutant plasmids. The change of the endogenous SUMOylation of PPARγ1 during M2 polarization was checked by IP and Western blotting by using PPARγ or SUMO1 antibodies in cell lysates of RAW264.7 cells and primary peritoneal macrophages induced by IL-4. The expression of some M2 related marker genes were detected by real-time quantitative polymerase chain reaction in PPARγ1-WT/mutants stably-overexpressed RAW264.7 cells. Chromatin immunoprecipitation (ChIP) experiment was used to confirm the different ability of binding to the promoter of arginase (Arg1) between PPARγ1-WT and PPARγ1-K77R. Results • It has been identified that the major SUMOylated site of PPARγ1 was Lys77, which could be de-SUMOylated by SENP1. The endogenous SUMOylation of PPARγ1 decreased when macrophage polarized to M2 macrophage induced by IL-4. The expression of Arg1 increased in PPARγ1-K77R stably-overexpressed RAW264.7 cells. PPARγ1-K77R easily bound to the promoter of Arg1 gene, showing more transcription activity. Conclusion • De-SUMOylation of PPARγ1 at Lys77 can enhance its transcription activity by promoting the expression of Arg1 gene, which is involved in the regulation of macrophage M2 polarization.

Effect of sumoylation on the structure and activity of human thymine DNA glycosylase / 上海交通大学学报（医学版）

Objective: To study the effect of sumoylation on the structure, stability and activity of human thymine DNA glycosylase (TDG). Methods: Expression and purification systems were established for obtaining SUMO-1-TDG protein with high purity which can be used for crystal screening and activity detection. Structure of SUMO-1-TDG was solved after crystal screening, diffraction data collection and structure analysis. The change of TDG stability led by sumoylation was detected through a protein thermal shift assay. In addition, an activity assay was applied to investigate the effect of sumoylation on the activity of TDG. Results: A high-resolution structure of SUMO-1-TDG which could clearly describe the interaction between TDG and SUMO-1 was solved. The melting temperature (Tm) value of SUMO-1-TDG increased by about 16℃ and the catalytic activity increased by 9.70%, comparing with TDG protein. Conclusion: SUMO-1 binds to TDG to modify the intermolecular interaction of amino acids near the binding site, and further participates in the regulation of the stability and catalytic activity of TDG protein.

SENP3-mediated de-SUMOylation of p53 inhibits its activity in human lung cancer cell lines / 上海交通大学学报（医学版）

Objective • To explore the effect of de-SUMOylation of p53 by SENP3 on its transcriptional activity in lung cancer. Methods • Lung cancer cell lines A549 (p53 wild type) and H1299 (p53 null) were used. Wild type p53 as well as sumoless mutants K386R or E388A were introduced into the cells. Co-immunoprecipitation was performed to detect SUMOylation of p53 under resting status or oxidative stress. Immunofluorescence was applied to observe the localization of p53 WT and K386R or E388A. Dual luciferase reporter assay and quantitative real-time PCR of p21 were performed to monitor the transcriptional activity of p53 WT and K386R or E388A. Growth curve was analyzed to demonstrate the effect of p53 WT and K386R or E388A on cell proliferation. Results • SENP3 was able to mediate de-SUMOylation modification of p53 under oxidative stress. Similar to WT p53, K386R and E388A p53 kept nuclear localization. Further, SENP3 knockdown or oxidative stress did not induce translocation of p53 from nucleus to cytoplasm. However, compared to WT, the transcriptional activation of K386R and E388A p53 were inhibited. Moreover, SENP3 inhibited the activity of p53 in A549. K386R and E388A p53 attenuated the inhibitive activity on cell proliferation in H1299. Conclusion • SENP3-mediated de-SUMOylation of p53 is one of mechanisms of its inactivation as tumor suppressor in lung cancer.

Ubiquitin-like modification (SUMOylation) and genomic stability / 上海交通大学学报（医学版）

The mechanism of protein post-translational modifications in regulation of cell mitosis and genomic stability is always a hot issue in biological researches. The present review introduced the functional activity of de-SUMOylase SENP3 in sister chromatin dissociation at mitosis, and demonstrated a new molecular mechanism of cross-talk regulation between protein SUMOylation and phosphorylation in maintaining genomic stability.

Effect of sumoylation on the structure and activity of human thymine DNA glycosylase / 上海交通大学学报（医学版）

Objective·To study the effect of sumoylation on the structure,stability and activity of human thymine DNA glycosylase (TDG).Methods·Expression and purification systems were established for obtaining SUMO-1-TDG protein with high purity which can be used for crystal screening and activity detection.Structure of SUMO-1-TDG was solved after crystal screening,diffraction data collection and structure analysis.The change of TDG stability led by sumoylation was detected through a protein thermal shift assay.In addition,an activity assay was applied to investigate the effect of sumoylation on the activity of TDG.Results·A high-resolution structure of SUMO-1-TDG which could clearly describe the interaction between TDG and SUMO-1 was solved.The melting temperature ? value of SUMO-1-TDG increased by about 16 ℃ and the catalytic activity increased by 9.70％,comparing with TDG protein.Conclusion·SUMO-1 binds to TDG to modify the intermolecular interaction of amino acids near the binding site,and further participates in the regulation of the stability and catalytic activity of TDG protein.

Progress of PML post-translational modifications in tumor cells / 中国病理生理杂志

The promyelocytic leukemia (PML) protein is an important part of the PML nuclear bodies (PML-NBs) structure.PML protein is crucial for the assembly of PML-NBs and recruits more than 30 different proteins, including DAXX, ATRX, and small ubiquitin-like molecules involving SUMO to the PML-NBs region.Increased evidence has emerged that a number of different proteins is involved in regulating PML activities by post-translational modifications, such as SUMO modification, ubiquitination and phosphorylation.Here, we review recent studies on the combination of PML and different proteins in the process of apoptosis, replicative senescence and DNA damage response.

SENP3 regulates the global protein turnover and the Sp1 level via antagonizing SUMO2/3-targeted ubiquitination and degradation

SUMOylation is recently found to function as a targeting signal for the degradation of substrates through the ubiquitin-proteasome system. RNF4 is the most studied human SUMO-targeted ubiquitin E3 ligase. However, the relationship between SUMO proteases, SENPs, and RNF4 remains obscure. There are limited examples of the SENP regulation of SUMO2/3-targeted proteolysis mediated by RNF4. The present study investigated the role of SENP3 in the global protein turnover related to SUMO2/3-targeted ubiquitination and focused in particular on the SENP3 regulation of the stability of Sp1. Our data demonstrated that SENP3 impaired the global ubiquitination profile and promoted the accumulation of many proteins. Sp1, a cancer-associated transcription factor, was among these proteins. SENP3 increased the level of Sp1 protein via antagonizing the SUMO2/3-targeted ubiquitination and the consequent proteasome-dependent degradation that was mediated by RNF4. De-conjugation of SUMO2/3 by SENP3 attenuated the interaction of Sp1 with RNF4. In gastric cancer cell lines and specimens derived from patients and nude mice, the level of Sp1 was generally increased in parallel to the level of SENP3. These results provided a new explanation for the enrichment of the Sp1 protein in various cancers, and revealed a regulation of SUMO2/3 conjugated proteins whose levels may be tightly controlled by SENP3 and RNF4.

Current situation and future development of SUMOylation in ophthalmology / 国际眼科杂志(Guoji Yanke Zazhi)

SUMOylation is a post - translational modification consisting of covalent conjugation of ubiquitin - like proteins called small ubiquitin related modifier ( SUMO ) . SUMO modification has been shown to significantly alter protein activity, which can modulate protein stability, affect protein-protein interactions, and modify protein localization and trafficking. This process adds another layer of control in eukaryote gene expression, and it regulates both transcriptional activation and repression. This article reviews the current situation and future development of SUMOylation in ophthalmology.

Relationship of RSUME and HIF-1α/VEGFA pathways with invasion of pituitary adenoma / 肿瘤

Objective: To investigate the relationships of RWD-containing sumoylation enhancer (RSUME) and hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway with invasion of pituitary adenoma. Methods: The expression levels of RSUME, HIF-1α and VEGF-A mRNAs in human pituitary adenoma tissues were detected by real-time fluorescence quantitative-PCR. The expression levels of small ubiquitin-related modifier-1 (SUMO-1), HIF-1α and VEGF-A proteins in pituitary adenoma tissues were detected by Western blotting. After transfecting with small interfering RNA (siRNA)-targeting RSUME into murine pituitary adenoma AtT-20 cells, the expression levels of RSUME, HIF-1α and VEGF-A mRNAs as well as SUMO-1, HIF-1α and VEGF-A proteins were detected by real-time fluorescence quantitative-PCR and Western blotting, respectively. The invasion ability was measured by Transwell assay. Results: The expression levels of RSUME, HIF-1α and VEGF-A mRNAs as well as SUMO-1, HIF- 1α and VEGF-A proteins in invasive pituitary adenoma tissues were significantly higher than those in non-invasive pituitary adenoma tissues and normal pituitary tissues (all P < 0.05). The SUMO-1 expression was positively correlated with HIF-1α expression in invasive pituitary adenoma tissues (r = 0.687, P < 0.05), and HIF-1α expression was positively correlated with VEGF-A expression (r = 0.773, P < 0.05). The expression levels of RSUME and VEGF-A mRNAs as well as SUMO-1, HIF-1α and VEGF-A proteins in AtT-20 cells after transfection with RSUME siRNA were significantly lower than those in the negative control group (AtT-20 cells transfected with negative control siRNA) and the blank control group (AtT-20 cells without any transfection) (all P < 0.05). The invasion ability of AtT-20 cells after transfection with RSUME siRNA was significantly inhibited (P < 0.05). Conclusion: RSUME can promote the invasion of pituitary adenoma by regulating HIF-1α/VEGF pathway. Silencing of RSUME gene may inhibit the invasion of pituitary adenoma cells.

Correlation between RSUME sumoylation and HIF-1α/VEGF pathway in gliomas / 西安交通大学学报(医学版)

Objective To explore the expressions and correlation of RWD containing sumoylation enhancer (RSUME),small ubiquitin-like modifier (SUMO-1),hypoxia inducible factor-1α(HIF-1α)and vascular endothelial growth factor (VEGF)in gliomas of different pathologic grade.Methods We investigated the expression levels of RSUME mRNA,HIF-1α mRNA and VEGF mRNA with reverse transcription-polymerase chain reaction (RT-PCR),and investigated the immunohistochemical staining to determine the expressions of SUMO-1,HIF-1α and VEGF in 63 cases of human gliomas of different pathologic grade and 9 cases of normal brain tissues.We studied its correlation with the pathologic grade and the relationship between the expression of RSUME promoter sumoylation and HIF-1α/VEGF pathway in gliomas.Results There were significant differences (P <0.01)in the expressions of RSUME mRNA,HIF-1αmRNA and VEGF mRNA in glioma tissues.With the increasing degree of pathologic grade in tumor specimens,the expression levels of RSUME mRNA,HIF-1α mRNA and VEGF mRNA increased markedly (P <0.01 ).There was a positive correlation of the expression levels of RSUME mRNA with HIF-1αmRNA and VEGF mRNA.There were significant differences (P <0.01 )in the expressions of SUMO-1,HIF-1αand VEGF in glioma tissues by immunohistochemical staining.With the ascending of pathologic grade of tumor specimens,the expression levels of SUMO-1,HIF-1α and VEGF increased markedly (P < 0.01 ).There was a positive correlation between the expression level of SUMO-1 and HIF-1α(r =0.857,P <0.01).The Kaplan-Meier analysis and Log-rank test showed significant differences in progress free survival (PFS)between the RSUME high-expression and low-expression groups (χ2 =36.032,P <0.01).Conclusion RSUME may enhance HIF-1α/VEGF pathway through sumoylation in gliomas.It implicates that RSUME is related to angiogenesis in gliomas and can promote tumor invasion and progression,indicating that RSUME can be a novel target in gliomas treatment.

Sumoylation of Hes6 Regulates Protein Degradation and Hes1-Mediated Transcription

BACKGROUND: Hes6 is a transcriptional regulator that induces transcriptional activation by binding to transcription repressor Hes1 and suppressing its activity. Hes6 is controlled by the ubiquitin-proteosome-mediated degradation system. Here we investigated the sumoylation of Hes6 and its functional role in its rhythmic expression. METHODS: Hes6, SUMO, and ubiquitin were transfected into HeLa cells and the expression pattern was observed by Western blot and immunoprecipitation. To confirm the effect of sumoylation on the rhythmic expression of Hes6, we generated mouse Hes6 promoter-driven GFP-Hes6 fusion constructs and expressed these constructs in NIH 3T3 cells. RESULTS: Overexpression of SUMO led to sumoylation of Hes6 at both lysine 27 and 30. Protein stability of Hes6 was decreased by sumoylation. Moreover, expression of a Hes6 sumoylation-defective mutant, the 2KR (K27/30R) mutant, or co-expression of SUMO protease SUSP1 with native Hes6, strongly reduced ubiquitination. In addition, sumoylation was associated with both the rhythmic expression and transcriptional regulation of Hes6. Wild type Hes6 showed oscillatory expression with about 2-hour periodicity, whereas the 2KR mutant displayed a longer period. Furthermore, sumoylation of Hes6 derepressed Hes1-induced transcriptional repression. CONCLUSION: Hes6 sumoylation plays an important role in the regulation of its stability and Hes1-mediated transcription. These results suggest that sumoylation may be crucial for rhythmic expression of Hes6 and downstream target genes.