Anti-tumor activities and apoptotic mechanism of ribosome-inactivating proteins

Ribosome-inactivating proteins(RIPs) belong to a family of enzymes that attack eukaryotic ribosomes and potently inhibit cellular protein synthesis.RIPs possess several biomedical properties,including anti-viral and anti-tumor activities.Multiple RIPs are known to inhibit tumor cell proliferation through inducing apoptosis in a variety of cancers,such as breast cancer,leukemia/lymphoma,and hepatoma.This review focuses on the anti-tumor activities of RIPs and their apoptotic effects through three closely related pathways:mitochondrial,death receptor,and endoplasmic reticulum pathways.

Crystallization and Preliminary Crystallographic Studies of Luffaculin 1,a Ribosome-inactivating Protein from the Seeds of Luffa Acutangula

Luffaculin 1 purified from the seeds of Luffa acutangula belongs to the type I ribosome-inactivating proteins （RIPs）. It has been crystallized by the vapor-diffusion method using polyethylene glycol as a precipitant. The crystal is of space group P1 with a = 39.135, b = 46.813, c = 83.571 A, α = 891068,β = 80.009 and y = 72.143°, and has two molecules per asymmetric unit. X-ray data have been collected to be 1.4 A using a synchrotron source.

Crystallization and Preliminary Crystallographic Studies of Cucurmosin 2,a Ribosome-inactivating Protein from the Sarcocarp of Cucurbita moschata

Cucurmosin 2, a type 1 ribosome-inactivating protein （RIP） isolated from sarcocarp of Cucurbita moschata, has been crystallized by the vapor-diffusion method using PEG6000 as the precipitant. The crystals belong to the orthorhombic space group P212121, with unit cell parameters a = 55.853, b = 65.507, c = 91.754 А, and have one molecule per asymmetric unit. X-ray data have been collected to 1.8А, using a synchrotron source.

苦瓜籽RIP的分离纯化及其对K562细胞作用的研究

目的:采用强阳离子交换层析柱(SP Sepharose H igh Perform ance)分离纯化苦瓜籽R IP,并观察苦瓜R IP对人红白血病细胞株K562细胞的抗肿瘤活性。方法:采用强阳离子交换层析柱(SP Sepharose H igh Perform-ance)分离纯化苦瓜籽R IP,SDS-PAGE、IEF等实验鉴定苦瓜籽R IP的纯度;以一定浓度梯度的苦瓜籽R IP处理K562细胞72 h,CCK-8检测其对细胞生长的影响;流式细细术(AnnexinⅤ)、细胞形态学(光镜及电镜)等方法检测苦瓜R IP诱导K562细胞凋亡的产生。结果:应用强阳离子交换层析柱(SP Sepharose H igh Perform ance)来分离纯化苦瓜籽R IP,可以快速方便分离得到高纯度的苦瓜R IP,得率也较高;一定作用剂量的苦瓜R IP不仅具有抑制K562细胞生长的活性(9.1μg/mL)的作用,还可诱导K562细胞发生凋亡。结论:运用强阳离子交换层析柱(SPSepharose H igh Perform ance)分离纯化苦瓜R IP是便捷可行的;苦瓜R IP具有抗肿瘤的活性,其作用可能是通过诱导细胞发生凋亡来实现。

RIP2的研究进展

受体相互作用蛋白（receptor—interacting protein．RIP）2属于RIP家族成员，在多种组织中均有分布。最初研究发现RIP2是Toll样受体（toll—lik ereceptors，TLRs）及NOD样受体（NOD—like receptors，NLRs）信号通路的下游因子，

Purification and characterization of Moschatin, a novel type Ⅰ ribosomeinactivating protein from the mature seeds of pumpkin (Cucurbita moschata), and preparation of its immunotoxin against human melanoma cells

A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of~29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC_(50) of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M_(21) with a IC_(50) of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.

Visualization of interaction between ribosome-inactivating proteins and supercoiled DNA with an atomic force microscope

The interaction between ribosome-inactivating proteins (RIPs) and supercoiled DNA was observed with an atomic force microscope (AFM). It was found that RIPs can bind to both supercoiled DNA and the unwound double stranded loop region in supercoiled DNA. The RIPs hound to the supercoils can induce the conformational change of supercoiled DNA. Furthermore, the supercoiled DNA was relaxed and cleaved into nick or linear form by RIPs. It indicated that RIP seemed to be a supercoil-dependent DNA binding protein and exhibited the activity of su-percoil-dependent DNA endonuclease.

构建MS2-RIP方法用于鉴定lncRNA DANCR在肿瘤细胞中的相互作用分子

【目的】为了探讨lncRNA DANCR在肿瘤细胞中的作用机制,本研究拟构建一种新的MS2-RIP方法用于鉴定DANCR的相互作用分子。【方法】利用MS2噬菌体衣壳蛋白可与噬菌体复制酶编码基因5’端一段由19个碱基组成的RNA茎环结构序列,即MS2结合位点(MS2 Binding Site,MS2 BS)高效结合的特点,构建串联表达MS2 BS和DANCR的过量表达质粒,通过MS2蛋白与MS2 BS的亲和作用,使DANCR得到富集,并且同步捕获与DANCR相互作用的分子,用于进一步分析鉴定。【结果】成功构建串联表达MS2 BS和DANCR的过量表达质粒用于MS2-RIP实验,该实验可显著富集DANCR以及作为阳性对照的DANCR结合蛋白EZH2。【结论】基于MS2蛋白与MS2结合位点的高效结合特性,我们针对lncRNA DANCR建立了MS2-RIP方法。该方法可有效富集DANCR以及与DANCR结合的生物活性分子,为研究DANCR的作用机制提供一种新的技术手段。

Trichobitacin-a new ribosome-inactivating protein Ⅰ.The isolation,physicochemical and biological properties of trichobitacin

From the press-residue of the fresh root tuber of Trichosanthes kirilowii Maxim (Cu-curbitaceae),a new ribosome-inactivating protein (RIP),trichobitacin,was isolated.It has the activity of RNA N-glycosidase and can inhibit the growth of human placental trophoblastic cells.Its molecular weight is 27,228 Da (ES-MS) and pI 9.6.It is a single chain basic RIP.Its amino acid composition was determined.It is a new RIP.It consists of 0.7~0.9% galactose and may be a glycoprotein.Its A'-and C-terminal amino acid is Asp and Ala,respectively.Its N-terminal preliminary amino acid sequence has been determined.

Primary structure of p-momorcharin, a ribosome-inactivating protein from the seeds of Momordica Charantia Linn (Cucurbitaceae)

The complete amino acid sequence of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae) has been determined. This has been done by the sequence analysis of peptides obtained by enzymatic digestion with trypsin, chymotrypsin and S. aureus V8 protease, as well as by chemical cleavage with BNPS-skatole. The protein consists of 249 amino acid residues containing one asparagine - linked sugar group attached to the site of Asn 5 1 and has a calculated relative molecular mass of 28,452 Da without addition of the carbohydrate. Comparison of this sequence with those of trichosanthin and other ribosome-inactivating proteins from different species of plants shows a significant homology with each other. Regarding the similarity of their biological properties, an active domain of these proteins has been predicted here.

植物类中药活性蛋白成分研究进展

从植物的蛋白质类成分中寻找新的药物是今后一个重要的发展方向。已有的研究表明 ,植物蛋白具有多方面的生物活性。核糖体失活蛋白是植物中广泛存在的一类具有较好抗肿瘤活性的蛋白质 ,通过干扰肿瘤细胞遗传信息链而诱导细胞凋亡 ;MAP30 ,PAP,GAP31等植物蛋白通过影响病毒 DNA复制表达而显示出抗 HIV病毒活性 ;多种植物蛋白还通过促进免疫细胞增殖、提高免疫细胞功能 ,起到增强免疫调节的作用。此外 ,在清除体内氧自由基、催化纤维蛋白原溶解等方面 ,植物蛋白也显示出明显的活性。

麻疯树(Jatropha curcas)毒蛋白curcin基因家族成员的分离和描述

麻疯树curcin基因家族中4个成员通过PCR扩增被分离.它们都不含有内含子,序列分析表明它们编码Ⅰ型核糖体失活蛋白(Ribosome-Inactivating Proteins,RIPs),证明curcin基因如同大多数RIPs一样由多基因家族编码.四个curcin基因家族成员和另两个已知curcin基因家族成员的开放阅读框(Open Reading Frame,ORF)的序列比对结果显示curcin基因家族成员至少可分为两个亚类群,亚类群之间的氨基酸序列相似性低于88%.分别将这4个curcin基因家族成员命名为curA2、curA3、curB2、curB3.对四条基因启动子区和ORF生物信息学分析推测curB2、curB3可能是被真菌及逆境诱导的curcin基因家族成员全长,而curA2、curA3可能是麻疯树胚乳贮存的curcin基因家族成员全长.Southern杂交结果显示,麻疯树基因组中至少有3个以上curcin基因家族成员.

苦瓜核糖体失活蛋白的纯化及其诱导HL-60细胞凋亡的研究

目的: 探讨运用假配基亲和层析和凝胶过滤层析相结合的手段纯化苦瓜核糖体失活蛋白(RIP)的可行性,同时观察其抑制HL-60细胞增殖并诱导其发生凋亡的生物学活性。方法: 利用假配基亲和层析法、凝胶过滤层析法提取纯化苦瓜RIP;通过绘制生长曲线和MTT检测观察苦瓜RIP抑制HL-60细胞增殖的活性,运用流式细胞仪检测细胞周期相分布及细胞凋亡率。结果: 运用硫酸铵分级沉淀、Blue Sepharose CL-6B假配基亲和层析、Sephadex-G75凝胶层析等方法提取、纯化得到了高纯度的苦瓜RIP,并且能够获得较高的得率;一定作用剂量的苦瓜RIP不仅能够抑制HL-60细胞的增殖活性,还可以诱导其发生凋亡。结论: 运用假配基亲和层析和凝胶过滤层析相结合的手段提取、纯化苦瓜RIP是可行的;苦瓜RIP可能是通过诱导细胞发生凋亡来发挥其抗肿瘤活性。

核糖体灭活蛋白在植物中的作用

植物核糖体灭活蛋白 (ribosome -inactivatingproteins ,RIPs)能够破坏真核或原核细胞的核糖体大亚基RNA ,使核糖体失活而不能与蛋白质合成过程中的延伸因子相结合 ,从而导致蛋白质合成受到抑制。不同的核糖体对不同RIPs的敏感性不同 ,RIPs对自体或异体核糖体的作用也有很大区别。RIPs对病毒有很强的抑制作用 ,并且有些RIPs表现出对某些真菌和昆虫的抗性 ,因此认为核糖体灭活蛋白在植物的防御反应中扮演重要角色。另外 ,RIPs还可能参与了细胞代谢、细胞死亡等生理调控过程。

苦瓜籽核糖体失活蛋白的基本性质研究

采用 Affi- Gel亲和层析和 Sephacryl S- 10 0分子筛层析等方法获得苦瓜籽核糖体失活蛋白 (RIP) ,经 SDS- PAGE,PAGE,IEF和 PAS方法分析均表明为单一蛋白着色带或单一糖蛋白着色带 .根据 SDS- PAGE测得表观分子量为 30 0 0 0 ,经 IEF- PAGE结果计算其 PI为 9.0 ,对无细胞系统中蛋白质生物合成抑制活性明显 ,其 IC50 为 5.3× 10 -10 mol/ L左右 .体外生物活性试验结果表明其对人肝癌细胞 ,Vero,SP2 / 0 ,3T3,Kb,Navana,S- 180等肿瘤细胞株和麻疹病毒均表现有不同程度的抑制作用 ,而对完整人胚肺二倍体细胞却毒性极小 .

天花粉蛋白基因的克隆及序列分析

本文应用ＤＮＡ多聚酶链式反应（ＰＣＲ）技术，从栝楼基因组ＤＮＡ中扩增并克隆了天花粉蛋白（ＴＣＳ）基因。核酸序列分析结果表明，克隆片段包括ＴＣＳ的前原蛋白的编码序列和５＇一侧翼区段。其编码序列与已发表的不同来源的３种ＴＣＳ基因的核苷酸序列的同源性分别为９９．２０％，９８．７４％和９８．６４％。推导出的氨基酸序列与已发表的４种ＴＣＳ的氨基酸序列的同源性分别为９８．６２％、９８．６２％、９７．４１％和９８．３８％。另外还发现，我们克隆的ＴＣＳ基因的５＇一侧翼区段比已发表的ＴＣＳ基因的相应区段多一个类似于ＴＡＴＡ盒的序列。我们进一步构建了该基因的一系列突变体，为深入研究ＴＣＳ基因结构、表达和调节机制以及ＴＣＳ的结构和功能关系奠定了重要的基础。

苦瓜核糖体失活蛋白生物活性与功能研究进展

核糖体失活蛋白(ribosome-inactivating proteins,RIPs)是一类能够脱去真核细胞28S r RNA内SRL区域的A4342,从而破坏延伸因子与核糖体的结合,将蛋白质的生物合成抑制在延伸阶段的蛋白质家族。RIPs有Ⅰ、Ⅱ、Ⅲ型,苦瓜中已发现的α-苦瓜素、β-苦瓜素、γ-苦瓜素、δ-苦瓜素、ε-苦瓜素、MAP30等,均属于Ⅰ型RIPs。这些RIPs具有抗病毒、抗菌、抗虫害、抑制肿瘤细胞生长等生物学活性,受到了人们的广泛关注。本文从RIPs的分类、生物学活性、功能与应用等方面,对苦瓜中的RIPs进行了综述。

美洲商陆抗病毒蛋白及其在艾滋病治疗中的应用

美洲商陆抗病毒蛋白是一种天然的广谱抗病毒试剂 ,其抗 HIV活性 ,已引起国内外学者的广泛兴趣和重视。旨在综述美洲商陆抗病毒蛋白的结构、核糖体失活活性、抗病毒活性及其在艾滋病治疗中的应用 。

蓖麻毒素及其解毒剂研究进展

蓖麻毒素(ricin)是一种从蓖麻籽中提取的剧毒植物蛋白,是核糖体失活蛋白(RIP)的一种,可以使核糖体失去蛋白质合成能力并最终导致细胞死亡,毒素在体内通过独特的"逆向转运"途径进入胞浆并与核糖体结合发挥毒性作用,目前对蓖麻毒素尚无有效的抗毒剂。本文对蓖麻毒素的结构,转运过程,作用机制以及抗毒剂的研究进展进行综述。

核糖体RNA拓扑学与RNAN－糖苷酶研究进展（上）

核糖体ＲＮＡ拓扑学的研究对阐明核糖体ＲＮＡ（ｒＲＮＡ）在蛋白质生物合成中的作用具有重要的意义。ＲＮＡＮ－糖苷０酶是一类核糖体失活蛋白．它只水解ｒＲＮＡ特定位置上一个腺苷酸的糖苷键，释放一个腺嘌呤碱基，使核糖体失活。ＲｉｃｉｎＡ链是研究得最早和最详细的ＲＮＡＮ－糖苷酶，迄今已发现有二十五种核糖体失活蛋白具有ＲＮＡＮ－糖苷酶活性。ＲＮＡＮ－糖苷酶作用于２８ＳｒＲＮＡ的α－ｓａｒｃｉｎ结构域，改变核糖体的构象而使其失活。