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;...     

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。     

miRNA中的G-四链体结构

G-四链体(G-quadruplex,G4)是由富含鸟嘌呤碱基的DNA或RNA序列组成的非典型核酸二级结构.在过去几十年中,人们着重研究了基因启动子区、UTR、端粒等常见基因功能区中的富G序列,探讨其结构与功能之间的关系.近些年,随着对非编码RNA在人类基因表达调控和疾病关系中的深入研究,非编码基因,尤其是miRNA中...     

G- 四链体DNA 结合剂研究进展

富含鸟嘌呤的单链DNA序列可以缠绕折叠形成G- 四链体结构。人类基因组中有36,000 个以上的DNA 序列有潜力生成G-四链体,如端粒末端重复序列,以及c-myc、c-kit、bcl-2 等原癌基因启动子区域。G-四链体是由四个鸟嘌呤之间通过Hoogsteen氢键形成G-四分体,相邻的G-四分体再通过π-π 堆积作用,由糖- 磷酸骨架相连而成。G- 四链体DNA 的形成有着重要的生物学意义,它和相关基因表达水平密切相关,诱导和稳定G- 四链体结构就有可能抑制癌基因的转录和表达,引起肿瘤细胞生物学功能的紊乱,从而抑制肿瘤细胞的增殖。G-四链体结构作为新的抗肿瘤药物靶点引起了科学家的广泛关注,能够稳定G- 四链体结构的配体包括二酰胺蒽醌类、苝类、阳离子卟啉类、金属配合物和天然产物等。本文对近年来以G-四链体为靶点的配体的研究进行了综述。     

G-四链体DNA结合剂研究进展

富含鸟嘌呤的单链DNA序列可以缠绕折叠形成G-四链体结构。人类基因组中有36,000个以上的DNA序列有潜力生成G-四链体,如端粒末端重复序列,以及c-myc、c-kit、bcl-2等原癌基因启动子区域。G-四链体是由四个鸟嘌呤之间通过Hoogsteen氢键形成G-四分体,相邻的G-四分体再通过π-π堆积作用,由糖-磷酸骨架相连而成。G-四链体DNA的形成有着重要的生物学意义,它和相关基因表达水平密切相关,诱导和稳定G-四链体结构就有可能抑制癌基因的转录和表达,引起肿瘤细胞生物学功能的紊乱,从而抑制肿瘤细胞的增殖。G-四链体结构作为新的抗肿瘤药物靶点引起了科学家的广泛关注,能够稳定G-四链体结构的配体包括二酰胺蒽醌类、苝类、阳离子卟啉类、金属配合物和天然产物等。本文对近年来以G-四链体为靶点的配体的研究进行了综述。     

G-四链体核酸的生物学功能

G-四链体是由富含鸟嘌呤的DNA或RNA折叠形成的高级结构。可形成G-四链体的序列在人基因组中广泛分布,涉及DNA复制、端粒维持、基因表达与调控以及遗传不稳定性等过程。研究发现有些化学合成的G-四链体序列也具有生物活性,如核仁素的核酸适体AS1411具有抑制恶性肿瘤增殖活性。G-四链体的生物学功能研究对于恶性疾病的发病机理和靶向治疗,以及设计开发核酸类抗癌药物有重要意义。     

G-四链体在卵巢癌治疗中的研究进展

卵巢癌(ovarian cancer, OC)是最常见的女性恶性肿瘤之一,缺乏有效的治疗手段是导致卵巢癌高死亡率的主要原因之一。DNA G-四链体(G4)是富含鸟嘌呤碱基的一种特殊核酸结构,该结构广泛存在于端粒、原癌基因启动子等区域,是目前抗肿瘤药物设计的重要靶点。此外,有研究表明,DNA G4可能还存在于炎症基因的启动子区域,进而参与炎症反应的调控。鉴于炎症反应在卵巢癌进展中的重要作用,DNA G4在卵巢癌治疗中可能具有抗肿瘤、抗炎的双重治疗效果。该文重点综述了DNA G4及其稳定剂在卵巢癌治疗中的最新研究进展。     

G-四链体结构的检测、功能及调控

G-四链体结构是近年来发现的特殊核酸二级结构,它在体内极易形成,分布十分广泛并且具有重要的生物学功能。研究者们已经在体外检测到G-四链体的存在并解析出其晶体结构,各种检测该结构的方法如特异性荧光探针、抗体等也不断被发现或合成。G-四链体不仅广泛分布于端粒、启动子区、外显子等具有重要功能的基因区域,在5'非编码区(5'UTR)、内含子区、3'非编码区(3'UTR)等也有广泛存在。相应区域的G-四链体参与到端粒延长、DNA复制、转录、减数分裂、基因重组等重要的生命过程,发挥抗肿瘤、抗病毒、抑制血管新生等作用。目前基于G-四链体结构的抗肿瘤药物已经进入临床试验阶段并取得了良好的疗效。G-四链体结构的内源性调节包括多种内源性蛋白以及碱基的甲基化等,维持其含量与结构的平衡状态。此外,外源性小分子也可对体内G-四链体的平衡状态发挥调节作用。本文将从化学、生物和医学的角度对G-四链体结构的检测方法及其特殊功能和调控进行系统的论述和展望。     

基因组G-四链体与疾病的关系

G-四链体(G-quadruplex,G4)是一种特殊的核酸二级结构,形成于单链DNA或者RNA的富含鸟嘌呤的区域.其广泛存在于基因组中,多位于端粒、启动子和UTR等区域.G4结构检测的生物物理学方法可以获取G4具体的空间结构信息,而生物学检测方法通常与高通量测序技术结合,可以对G4做全基因组范围内的测序分析.G4结构...     

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。     

A fine-tuning mechanism underlying self-control for autophagy: deSUMOylation of BECN1 by SENP3

ABSTRACT

The roles of SUMOylation and the related enzymes in autophagic regulation are unclear. Based on our previous studies that identified the SUMO2/3-specific peptidase SENP3 as an oxidative stress-responsive molecule, we investigated the correlation between SUMOylation and macroautophagy/autophagy. We found that Senp3^± mice showed increased autophagy in the liver under basal and fasting conditions, compared to Senp3^+/+ mice. We constructed a liver-specific senp3 knockout mouse; these Senp3-deficient liver tissues showed increased autophagy as well. Autophagic flux was accelerated in hepatic and other cell lines following knockdown of SENP3, both before and after the cells underwent starvation in the form of the serum and amino acid deprivation. We demonstrated that BECN1/beclin 1, the core molecule of the BECN1-PIK3C3 complex, could be SUMO3-conjugated by PIAS3 predominantly at K380 and deSUMOylated by SENP3. The basal SUMOylation of BECN1 was increased upon cellular starvation, which enhanced autophagosome formation by facilitating BECN1 interaction with other complex components UVRAG, PIK3C3 and ATG14, thus promoting PIK3C3 activity. In contrast, SENP3 deSUMOylated BECN1, which impaired BECN1-PIK3C3 complex formation or stability to suppress the PIK3C3 activity. DeSUMOylation of BECN1 restrained autophagy induction under basal conditions and especially upon starvation when SENP3 had accumulated in response to the increased generation of reactive oxygen species. Thus, while reversible SUMOylation regulated the degree of autophagy, SENP3 provided an intrinsic overflow valve for fine-tuning autophagy induction.     

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.     

Effects of cation charges on the binding of stabilizers with human telomere and TERRA G-quadruplexes

Both telomere and telomeric repeat-containing RNAs (TERRA) can fold into G-quadruplexes (G4) in eukaryotic cells. Given their key roles in the regulation of telomere length and translation, telomere and TERRA G4 are interesting targets of novel drug development strategies. It is known that the cation charge of a stabilizer is crucial to the binding of G4 and stabilizer. However, the quantitative relationship between the cation charge of a stabilizer and the binding strengths with telomere and TERRA G4 remain unclear. In the current study, by substituting positive charged TMPyP4 with neutral and negative charged groups, the effects of cation charges on the binding conformation and binding strength of porphyrin stabilizers are investigated via molecular docking and molecular dynamic (MD) simulations. The results show that all TMPyP4 analogs form stable binding complexes with telomere and TERRA G4 and that, stabilizer charges have limited effects on binding conformation and can hardly lead to any special conformational alternations of G4. Our hydrogen bond analysis shows that all stabilizers can hardly form stable intermolecular hydrogen bonds with G4. Regarding binding strength levels, a linear correlation is found between the binding free energies and cation charges of stabilizers in all G4?stabilizer complexes, revealing the pivotal role of electrostatic interactions. The present work is the first to reveal a quantitative correlation between the charges and binding strengths of stabilizers in their binding with human telomere and TERRA G4, which will prove pivotal for G4 targeted drug design and development.     

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.     

Molecular Cloning and Bacterial Expression of the Catalytic Domain of the SENP1 Gene of Oryzias latipes

In this study, we cloned the catalytic domain of the Oryzias latipes sentrin/SUMO-specific protease 1 (OlSENP1-CD) gene and produced the recombinant OlSENP1-CD protein in Escherichia coli. Experimental procedures designed to reveal the ability of the recombinant protein to show deSUMOylating activity in vitro should be helpful in future studies of other SENPs and the SUMO pathway.     

Stereospecific activity of 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine and comparison of analogs in the degranulation of platelets and neutrophils

1-O-Hexadecyl-2-O-acetyl-$\text{sn}$-glycero-3-phosphocholine (platelet activating factor) stimulated the degranulation of rabbit platelets and human neutrophils, whereas the enantiomer, 3-O-hexadecyl-2-O-acetyl-$\text{sn}$-glycero-1-phosphocholine, was inactive. The analogs compared had the following relative potencies in degranulating platelets and neutrophils: 1-O-hexadecyl-2-O-acetyl-$\text{sn}$-glycero-3-phosphocholine > 1-O-hexadecyl-2-O-ethyl-$\text{sn}$-glycero-3-phosphocholine >$\text{rac}$-1-O-octadecyl-2-O-ethylglycero-3-phosphocholine = 1-O-hexadecyl-2-O-methyl-$\text{sn}$-glycero-3-phosphocholine >$\text{rac}$-1-O-dodecyl-2-O-ethyl-glycero-3-phosphocholine. The deacetylated compound, 1-O-hexadecyl-2-lyso-$\text{sn}$-glycero-3-phosphocholine, and 1-O-hexadecyl-2,2-dimethylpropanediol-3-phosphocholine were inactive. The active analogs selectively desensitized the response to each other in the neutrophils. It is suggested that these compounds may activate cells through interaction with a stereospecific receptor.