基于单细胞测序探索SENP2在肾细胞癌不同亚型中的作用

本研究基于单细胞测序数据探讨SENP2在肾细胞癌(renal cell carcinoma, RCC)3种不同亚型中的表达差异和作用机制。通过R语言进行UMAP降维可视化和差异表达基因(differentially expressed genes, DEGs)的筛选;对DEGs进行功能注释和富集分析并构建蛋白相互作用(protein-protein interaction, PPI)网络,通过Cytoscape软件筛选关键基因;通过分析SENP2的表达差异与3种RCC亚型患者生存预后和临床分期的关系,揭示其在不同亚型中的作用;通过分析SENP2及其互作基因的相关性,探索SENP2引起3种不同RCC亚型患者生存预后和临床分期差异性的调控机制;最后,对肾透明细胞癌(kidney renal clear cell carcinoma, KIRC)中的SENP2进行免疫组化实验。本研究结果表明,RCC 3种亚型中的基因表达具有差异性;RCC 3种亚型中差异表达基因显著富集于细胞增殖、蛋白质代谢、细胞衰老以及TP53调节等生物学过程中;筛选关键基因,锁定SENP2;生存分析和临床分期分析结果表明...     

SENP1在大鼠低氧性肺动脉高压形成中肺血管壁中表达及可能作用

目的:探讨大鼠低氧性肺动脉高压(HPH)形成过程中SENP1在肺小动脉的动态表达变化及作用。方法:40只成年雄性Wistar大鼠随机分为5组(n=8):对照组和缺氧3 d、7 d、14 d2、1 d组,常压间断低氧复制HPH大鼠模型。测各组大鼠平均肺动脉压(mPAP)、右心室肥大指数(RVHI)、血管形态学指标;原位杂交、逆转录-聚合酶链反应(RT-PCR)检测肺内SUMO特异性蛋白酶-1(SUMO-specific proteases-1,SENP1)mRNA表达,免疫组化、Westernblot检测其蛋白质水平。结果:①缺氧7 d后,肺小动脉出现血管重塑,且mPAP明显上升;低氧14 d后,肺小动脉重塑更明显,mPAP达高峰。RVHI在低氧14 d后明显增加。②原位杂交显示,SENP1 mRNA在对照组肺小动脉壁呈阳性表达,低氧后其相对量无明显变化。RT-PCR显示肺组织SENP1 mRNA表达与原位杂交所观察到的肺小动脉壁SENP1 mRNA变化趋势一致;SENP1蛋白在对照组呈阳性表达,低氧7 d后其表达量开始呈进行性下降。Western blot显示肺组织内SENP1蛋白表达与免疫组化观察到的肺小动脉壁SENP1蛋白变化趋势一致。③SENP1蛋白与mPAP、重塑指数、RVHI均呈负相关。结论:慢性低氧诱导肺小动脉壁SENP1蛋白降解,进而可能在HPH发病过程中发挥一定的作用。     

HIF-1α的可逆性SUMO化修饰

低氧诱导因子1(hypoxia inducible factor-1, HIF-1)是参与调节机体氧平衡的重要转录因子,在细胞低氧应答反应中起核心作用,能调节100多种涉及低氧应激下细胞适应和存活的靶基因．HIF-1由氧敏感的α亚基和在细胞内稳定表达的β亚基组成．其中α亚基可受到多种翻译后化学修饰作用,如在常氧下,HIF-1α通过泛素化蛋白酶修饰并导致其快速降解．最近几年发现的泛素样蛋白家族成员小泛素蛋白样修饰蛋白（SUMO）也能与HIF-1α共价结合．SUMO是一种分子量约为12 kD的小蛋白,从拟南芥到人类普遍存在．SUMO可共价结合许多靶底物蛋白,并对其进行翻译后修饰,该过程称为SUMO化．与泛素化蛋白酶体途径不同的是,SUMO化修饰能在常氧和相对低氧的条件下调节HIF-1α蛋白的稳定性,从而改变其转录活性．SUMO化是一个可逆的动态过程,可被特异性蛋白酶ULP/SENP将其从底物上去除．本文主要就HIF-1α的可逆性SUMO化修饰作一综述．     

小泛素相关修饰物SUMO研究进展

蛋白质翻译后修饰对改变蛋白功能、活性或定位都起着非常重要的作用,泛素及其相似蛋白的修饰是其中一种重要形式。与其他诸如磷酸化、乙酰化、糖基化等不同的是,泛素及其相似蛋白的修饰基团本身即是一个小的多肽,通过异肽键与靶蛋白Lys侧链ε-NH2相连,其中小泛素相关修饰物(small ubiquitin—related modifier,SUMO)与蛋白的共价连接是一种新的广泛存在的翻译后修饰形式。SUMO是广泛存在于真核生物中高度保守的蛋白家族,在脊椎动物中有三个SUMO基因,称为SUMO-1,-2,-3,与泛素在二级结构上极其相似,且催化修饰过程的酶体系也具有很高的同源性。然而,与泛素化介导的蛋白酶降解途径不同,SUMO化修饰发挥着更为广泛的功能,如核质转运、细胞周期调控、信号转导、转录活性调控等。     

去甲基化酶JARID1D在前列腺癌侵袭和转移中的作用与机制

目的 探索去甲基化酶JARID1D在前列腺癌侵袭和转移中的作用与机制,以期为前列腺癌的转移防治提供新的预测指标及治疗靶点。方法 选择前列腺癌细胞系22RV1和DU145转染慢病毒,以敲低JARID1D的表达,获得细胞系sh-JARID1D,并通过qRT-PCR及Western Blot检测转染后22RV1和DU145细胞中JARID1D的表达情况。通过Transwell实验检测JARID1D低表达后细胞侵袭能力的变化。通过裸鼠尾静脉注射sh-JARID1D细胞系建立体内实验转移模型,观察敲低JARID1D表达后体内肿瘤细胞的转移能力的变化。通过qRT-PCR及Western Blot检测JARID1D对转移相关基因基质金属蛋白酶2(MMP2)表达的影响。结果 Transwell实验显示JARID1D低表达的前列腺癌细胞侵袭能力增强(22RV1细胞系P<0.01,DU145细胞系P<0.001);体内实验显示JARID1D低表达可显著增强肿瘤细胞的体内转移潜能(22RV1转移模型P<0.01,DU145转移模型P<0.01)。进一步实验表明降低JARID1D表达,...     

蛋白质SUMO化修饰与肿瘤调控

SUMO化修饰是一种重要的蛋白质翻译后修饰方式,在细胞周期调控、细胞代谢、基因转录、DNA损伤和修复等众多细胞生物学过程中,对底物蛋白质的表达、定位和活性进行调控。蛋白质SUMO化修饰是动态可逆的过程,去SUMO化修饰由SUMO特异性蛋白酶(SENPs)家族成员所催化。由于受到SUMO化修饰的底物蛋白种类众多、功能多样,SUMO化修饰能够在整体和特定蛋白质修饰层面,参与调控肿瘤的发生发展,并且这种调控机制非常复杂,比如调控细胞周期的进程、DNA损伤和基因组不稳定性、肿瘤代谢与生长、抗肿瘤免疫等。SENPs家族成员是底物蛋白质SUMO化修饰程度的决定者,该研究团队对SENPs家族成员在肿瘤中的作用开展了系列研究,因此该文也将以SENP1和SENP3为例,对SENPs在肿瘤进程中的作用及其作用机制展开介绍。     

大鼠SUMO特异性蛋白酶1催化区蛋白制备及功能鉴定*

目的: 制备大鼠SUMO特异性蛋白酶1(sentrin-specific protease,SENP1)催化结构域(SENP1C)蛋白,并鉴定其酶活性。方法: 分别以大鼠SENP1-pcDNA3.1和EGFP-pcDNA3.1重组体为模板,PCR扩增目的基因,克隆入pGEM-T载体;酶切鉴定后,再亚克隆入原核表达载体pET-28a;阳性重组体导入原核表达细胞BL-21,异丙基硫半乳糖苷(IPTG)诱导蛋白质表达;SDS-PAGE及考马斯亮蓝染色鉴定蛋白质的表达。Ni-NTA吸附纯化蛋白质并透析处理,SDS-PAGE及考马斯亮蓝染色鉴定蛋白质的纯度;1 μmol/L及5 μmol/L Tat-EGFP分别孵育HT22细胞不同时间,荧光显微镜下观察细胞转染情况。采用5 μmol/L Tat-SENP1C预孵育HT22细胞10 h,免疫印迹检测整体蛋白质的SUMO化水平;用5 μmol/L Tat-SENP1C预孵育HT22细胞或过表达Myc-Akt1和HA-SUMO1的HT22细胞10 h后,免疫沉淀和免疫印迹检测内源性和外源性Akt1与SUMO1的结合(SUMO化)。结果: Tat-SENP1C-pET-28a和Tat-EGFP-pET-28a重组原核表达载体成功构建,IPTG可以诱导蛋白质高表达;采用Ni-NTA纯化和透析可获得较高纯度的蛋白质;5 μmol/L Tat-EGFP孵育HT22 细胞10 h后,蛋白质穿膜效率较高;Tat-SENP1C重组蛋白可以显著降低HT22细胞中整体蛋白质的SUMO化以及内源性和外源性Akt1 SUMO化。结论: Tat-SENP1C-pET-28a和Tat-EGFP-pET-28a重组原核表达载体构建成功,且被IPTG诱导后可高效表达蛋白质;纯化的Tat-SENP1C蛋白具有较强的穿膜能力及酶活性。     

植物SIZ1 SUMO E3连接酶的研究进展

SUMO化修饰是一种重要的翻译后修饰,对蛋白的翻译后调控起到重要作用。植物SIZ1是一种SUMOE3连接酶,在SUMO化的过程中起着关键作用。本文概述了SIZ1的基本结构和功能,阐述了其在植物响应非生物胁迫如高温、低温、干旱、盐和离子胁迫时所发挥的调节功能,并展望了植物SIZ1研究中有待解决的问题。     

泛素化和SUMO化对DEC1的拮抗调控作用

分化的胚软骨表达蛋白1(differentiated embryo-chondrocyte expressed gene 1,DEC1)作为一种时钟蛋白,除了在周期节律的调控中发挥转录抑制作用外,还在能量代谢以及多种肿瘤相关的信号通路的调控中发挥重要作用。此外,蛋白质的翻译后修饰是实现蛋白质功能精细调控的一种重要方式。目前发现,DEC1主要可被两种翻译后修饰,即泛素化和SUMO化修饰。尽管泛素化和SUMO化是两种过程非常类似的蛋白质翻译后修饰方式,但是它们对目的蛋白功能的调控却截然不同。由于泛素化和SUMO化与底物的作用靶点都是赖氨酸(Lys),因此在多数情况下,泛素化和SUMO化以拮抗性的方式调控底物蛋白的功能。鉴于此,该文旨在阐述泛素化和SUMO化修饰对DEC1功能的拮抗调节过程,为了解时钟蛋白DEC1对多种信号通路的调控过程中的分子机制提供新的思路。     

PC-1在前列腺癌细胞中促进c-myc基因的表达

前列腺癌相关基因PC-1(Prostate and colon gene1)是属于癌基因D52家族成员,具有促进前列腺癌细胞雄激素非依赖性生长的功能。为了研究PC-1发挥这种生物功能的分子机制,文章在PC-1高表达的LNCaP-pc-1及对照LNCaP-zero细胞中,利用RT-PCR和Western blotting等方法检测c-myc基因表达;提取两细胞胞质和胞核蛋白,利用Western blotting分析c-myc上游调节蛋白β-catenin变化;利用c-Myc蛋白抑制剂10058-F4作用前列腺癌细胞C4-2,Western blotting检测PC-1蛋白表达变化。发现PC-1促进c-myc基因表达,并促进β-catenin入核;c-Myc蛋白抑制剂10058-F4可抑制PC-1的表达。结果表明:PC-1在前列腺癌中促进c-myc基因的表达,并且这种促进作用可能是通过Wnt/β-catenin信号通路实现的。同时,PC-1与c-Myc蛋白间可相互促进,进一步促进前列腺癌细胞雄激素非依赖性生长。     

The SUMO protease SENP3 regulates mitochondrial autophagy mediated by Fis1

Mitochondria are unavoidably subject to organellar stress resulting from exposure to a range of reactive molecular species. Consequently, cells operate a poorly understood quality control programme of mitophagy to facilitate elimination of dysfunctional mitochondria. Here, we used a model stressor, deferiprone (DFP), to investigate the molecular basis for stress‐induced mitophagy. We show that mitochondrial fission 1 protein (Fis1) is required for DFP‐induced mitophagy and that Fis1 is SUMOylated at K149, an amino acid residue critical for Fis1 mitochondrial localization. We find that DFP treatment leads to the stabilization of the SUMO protease SENP3, which is mediated by downregulation of the E3 ubiquitin (Ub) ligase CHIP. SENP3 is responsible for Fis1 deSUMOylation and depletion of SENP3 abolishes DFP‐induced mitophagy. Furthermore, preventing Fis1 SUMOylation by conservative K149R mutation enhances Fis1 mitochondrial localization. Critically, expressing a Fis1 K149R mutant restores DFP‐induced mitophagy in SENP3‐depleted cells. Thus, we propose a model in which SENP3‐mediated deSUMOylation facilitates Fis1 mitochondrial localization to underpin stress‐induced mitophagy.     

Structural insights into the SENP6 Loop1 structure in complex with SUMO2

The SENP proteases regulate the SUMO conjugates in the cell by cleaving SUMO from target proteins. SENP6 and SENP7 are the most divergent members of the SENP/ULP protease family in humans by the presence of insertions in their catalytic domains. Loop1 insertion is determinant for the SUMO2/3 activity and specificity on SENP6 and SENP7. To gain structural insights into the role of Loop1, we have designed a chimeric SENP2 with the insertion of Loop1 into its sequence. The structure of SENP2‐Loop1 in complex with SUMO2 was solved at 2.15 Å resolution, and reveals the details of an interface exclusive to SENP6/7 and the formation of unique contacts between both proteins. Interestingly, functional data with SUMO substrates showed an increase of the proteolytic activity in the SENP2‐Loop1 chimera for diSUMO2 and polySUMO2 substrates.     

SENP3‐mediated deSUMOylation of dynamin‐related protein 1 promotes cell death following ischaemia

Global increases in small ubiquitin‐like modifier (SUMO)‐2/3 conjugation are a neuroprotective response to severe stress but the mechanisms and specific target proteins that determine cell survival have not been identified. Here, we demonstrate that the SUMO‐2/3‐specific protease SENP3 is degraded during oxygen/glucose deprivation (OGD), an in vitro model of ischaemia, via a pathway involving the unfolded protein response (UPR) kinase PERK and the lysosomal enzyme cathepsin B. A key target for SENP3‐mediated deSUMOylation is the GTPase Drp1, which plays a major role in regulating mitochondrial fission. We show that depletion of SENP3 prolongs Drp1 SUMOylation, which suppresses Drp1‐mediated cytochrome c release and caspase‐mediated cell death. SENP3 levels recover following reoxygenation after OGD allowing deSUMOylation of Drp1, which facilitates Drp1 localization at mitochondria and promotes fragmentation and cytochrome c release. RNAi knockdown of SENP3 protects cells from reoxygenation‐induced cell death via a mechanism that requires Drp1 SUMOylation. Thus, we identify a novel adaptive pathway to extreme cell stress in which dynamic changes in SENP3 stability and regulation of Drp1 SUMOylation are crucial determinants of cell fate.     

The deSUMOylase SENP7 promotes chromatin relaxation for homologous recombination DNA repair

SUMO conjugation is known to occur in response to double‐stranded DNA breaks in mammalian cells, but whether SUMO deconjugation has a role remains unclear. Here, we show that the SUMO/Sentrin/Smt3‐specific peptidase, SENP7, interacts with the chromatin repressive KRAB‐associated protein 1 (KAP1) through heterochromatin protein 1 alpha (HP1α). SENP7 promotes the removal of SUMO2/3 from KAP1 and regulates the interaction of the chromatin remodeler CHD3 with chromatin. Consequently, in the presence of CHD3, SENP7 is required for chromatin relaxation in response to DNA damage, for homologous recombination repair and for cellular resistance to DNA‐damaging agents. Thus, deSUMOylation by SENP7 is required to promote a permissive chromatin environment for DNA repair.     

The IL-6 family of cytokines modulates STAT3 activation by desumoylation of PML through SENP1 induction

Post-translational modification by small ubiquitin-like modifier (SUMO) plays an important role in the regulation of different signaling pathways and is involved in the formation of promyelocytic leukemia (PML) protein nuclear bodies following sumoylation of PML. In the present study, we found that IL-6 induces desumoylation of PML and dissociation between PML and SUMO1 in hepatoma cells. We also found that IL-6 induces mRNA expression of SENP1, a member of the SUMO-specific protease family. Furthermore, wild-type SENP1 but not an inactive SENP1 mutant restored the PML-mediated suppression of STAT3 activation. These results indicate that the IL-6 family of cytokines modulates STAT3 activation by desumoylation and inactivation PML through SENP1 induction.     

SENP1启动子G-四链体鉴定及其作用研究

目的:研究G-四链体(G4)对SUMO特异性蛋白酶1(SUMO-specific proteases 1,SENP1)基因的转录调控作用。方法:克隆不同的SENP1启动子片段构建SENP1启动子报告质粒,通过报告基因检测鉴定SENP1启动子核心转录调控区;分析SENP1启动子核心转录调控区序列,并进行G4形成序列预测;合成G4形成序列寡核苷酸,利用圆二色谱分析检测G4形成序列寡核苷酸的拓扑结构;通过G4配体TMPyP4处理和过表达G4解旋酶G4R1结合报告基因检测和Western blot鉴定启动子G4对SENP1转录表达的调控作用。结果:发现-910～+226区域是SENP1启动子的核心转录调控区,序列富含G/C;生信分析发现SENP1启动子核心区存在G4形成序列;圆二色谱分析证实SENP1启动子G4形成序列能够形成G4结构;报告基因检测和Western blot检测发现启动子G4对SENP1转录表达具有抑制作用。结论:SENP1启动子核心转录调控区存在G4结构并对其转录表达具有抑制作用,为揭示SENP1在生理和病理过程中的作用机制提供新的研究思路和试验线索。     

SENP1-mediated NEMO de-SUMOylation inhibits intermittent hypoxia induced inflammatory response of microglia in vitro

Among the seven small ubiquitin-like modifier (SUMO)-specific proteases (SENPs), our previous work showed that SENP1 suppressed nuclear factor kappa B (NF-κB) activation and alleviates the inflammatory response in microglia. However, the mechanism is still largely unknown. In this study, western blot analysis and enzyme-linked immunosorbent assay were utilized for evaluating the extent of NF-κB activation and expression of proinflammatory cytokines. qPCR and western blot analysis were performed to detect SENP1 expression. Coimmunoprecipitation followed by western blot analysis was applied to measure the changes in SUMOylation of NF-κB essential modulator (NEMO) and P65 in microglia with or without overexpression of SENP1. As the results, we found that intermittent hypoxia (IH) triggered the activation of NF-κB and upregulated the expression levels of tumor necrosis factor-α and interleukin-6. Interestingly, our data indicated that the SUMOylation of NEMO was enhanced by IH while SUMOylation of P65 was not affected. Further, our data showed that overexpression of SENP1 could decrease the extent of NF-κB activation and inhibit the inflammatory response of microglia through regulating the SUMOylation of NEMO. Collectively, this study presents the first report of the SENP1-controlled de-SUMOylation process of NEMO and its critical role in regulating NF-κB activation and proinflammatory cytokines secretion in microglia cells. This study would benefit for clarifying the role of SENP1 in IH-induced activation of microglia, thus providing potential therapeutic targets for obstructive sleep apnea treatment.