核糖体失活蛋白的结构功能与分布

核糖体失活蛋白是一类在植物中较广泛存在的毒蛋白。植物核糖体失活蛋白具有RNAN-糖苷酶活力,可作用于核糖体RNA,使核糖体失去蛋白质合成的功能。根据一级结构,核糖体失活蛋白可分为两种类型。Ⅰ型核糖体失活蛋白由一条链组成,分子量在25—30 kDa之间。Ⅱ型核糖体失活蛋白由两条以二硫键相连的链(A、B链)组成,分子量在60 kDa左右。B链可以与细胞表面含半乳糖的受体结合,有助于A链进入细胞,作用于核糖体。目前至少已从9个科31种植物中分离纯化了Ⅰ型RIP。Ⅱ型RIP较少,仅在6科8种植物中发现。除了具有RNA N-糖苷酶活性,还发现一些核糖体失活蛋白可以切割超螺旋双链DNA,产生缺口环状和线状DNA。此外,一种Ⅰ型RIP,克木毒蛋白还具有超氧化物歧化酶活性。     

核糖体RNA拓扑学与RNA N－糖苷酶研究进展（下）

28S rRNA的α-sarcin结构域直接参与核糖体催化的蛋白质合成反应,已经证明天花粉蛋白是一种RNA N-糖苷酶,一种测定RNA N-糖苷酶活力的新方法也已初步建立.天花粉蛋白能使超螺旋DNA解旋并断裂为缺口环状和线状DNA.并已发现其它RNA N-糖苷酶也具有这一核酸内切活性.天花粉蛋白对28S rRNA,超螺旋DNA和艾滋病毒(HIV-1)RNA三种底物可能有相同的分子作用机制.     

放射性同位素标记测定RNA N-糖苷酶活性的新方法

核糖体失活蛋白(ribosome-inactivating protein,RIP)是一类抑制真核细胞蛋白质生物合成的毒蛋白。近几年发现,有相当一部分RIP的作用机制属于RNAN-糖苷酶(RNA NGlycosidase),如蓖麻毒蛋白A链(ricinA-chain)及一些单链RIP。它们能专一水解大鼠核糖体28 SRNA的第4324位腺苷酸的C-N糖苷键,释放一个腺嘌呤碱基,在相应的核糖Cl位上留下一个醛基。     

几种核糖体失活蛋白对超螺旋环状DNA的切割与解旋作用

天花粉蛋白(trichosanthin)是一种单链核糖体失活蛋白(ribosome-inactivating protein,RIP),它失活核糖体的机制属于RNA N-糖苷酶型。最近Li等发现天花粉蛋白可作用于超螺旋环状DNA,将其切割与解旋成缺口及线状分子,但并不作用于线状DNA。为了     

SUMO化修饰的Apak对核糖体RNA合成的调控作用

核糖体是由核糖体RNA和核糖体蛋白组成的复合体,其功能是参与蛋白质合成.SUMO化修饰的底物蛋白对核糖体的形成有重要调控作用.前期研究发现,KRAB型锌指蛋白Apak能特异地抑制p53所介导的凋亡通路.进一步研究发现,在核仁应激及癌基因激活条件下,抑癌蛋白ARF促进Apak发生SUMO化修饰并促使其移位于核仁.为了进一步探讨SUMO化修饰的Apak对核糖体RNA合成的调控功能,本研究通过Northern blot检测SUMO化修饰的Apak对核糖体RNA合成的影响,实时定量PCR检测核糖体RNA转录水平,RNA-Ch IP方法检测核糖体RNA与Apak蛋白的相互作用,结果表明,SUMO化修饰的Apak抑制47S核糖体RNA前体的合成且抑制RNA聚合酶Ⅰ介导转录的18S和5.8S r RNA的合成;在放线菌素D以及癌基因诱导下,促进Apak与18S,5.8S r RNA相互作用.本研究对理解Apak的功能和作用机制提供了新的依据,为深入研究KRAB型锌指蛋白家族分子对核糖体RNA的调控奠定了基础.     

核糖体失活蛋白与超螺旋DNA相互作用的原子力显微镜直接观察

运用原子力显微镜研究了核糖体失活蛋白 (RIP)与超螺旋DNA相互作用 ,发现这类毒蛋白 (克木毒蛋白和蓖麻毒蛋白A链 ) ,既能与超螺旋形式的DNA结合 ,又能结合在超螺旋DNA分子中未解旋的双链环区 .在与超螺旋DNA结合后 ,引起超螺旋DNA构象变化以利解旋并与双链DNA结合 ,进而将松弛的DNA双链切成缺口或线性形式 .这说明RIP是一种超螺旋DNA结合蛋白 ,并表现出依赖超螺旋的DNA内切酶活性     

快速检测核糖体失活蛋白活性质粒的构建及应用

核糖体失活蛋白专一地断裂28S rRNA第4 324位的腺嘌呤与核糖之间的N-糖苷键,具有特异破坏核糖体的结构,抑制蛋白质生物合成的功能。核糖体失活蛋白在医疗方面有极大的应用价值。为了能简单快速筛选出核糖体失活蛋白,本实验构建了一种包含核糖体失活蛋白识别位点的双荧光素酶质粒psiCHECKTM-2-F28RNA。用具有N 糖苷酶活性的苦荞凝集素(tartary buckwheat lectin,TBL)作用于psiCHECKTM-2-F28RNA质粒,电泳检测发现,TBL可以将质粒DNA由超螺旋型切割为缺刻型。将psiCHECKTM-2-F28RNA转染HCT116细胞,发现海肾/萤火虫荧光比值也明显降低,表明构建的质粒可以用于检测核糖体失活蛋白对细胞的毒性作用。当将psiCHECKTM-2-F28RNA中的GAGA序列中腺嘌呤分别突变后进行同样实验,确定该质粒中的GAGA为核糖体失活蛋白的识别位点。进一步构建包含GAGA特征序列的Wnt1-3′UTR区的质粒psiCHECKTM-2-Wnt1-3′UTR,实验也发现,在胞外和胞内TBL与psiCHECKTM-2-Wnt1-3′UTR都具有相互作用,表明细胞内具有GAGA序列的mRNA也可能成为核糖体失活蛋白的靶点。选用几种食源性作物中提取的蛋白质,分别与psiCHECKTM-2-F28RNA作用,进行体外检测,结果显示,该质粒能快速地筛选来源于不同生物的核糖体失活蛋白。这些结果表明,本实验构建的psiCHECKTM-2-F28RNA质粒,可用于核糖体失活蛋白的快速筛选和酶活性鉴定。     

天花粉蛋白的作用机制—RNA N—糖苷酶型

核糖体失活蛋白(ribosome-inactivating protein,RIP)是一类广泛分布于植物界的能抑制真核细胞核糖体功能的毒蛋白。其作用的分子机制有两类:(1)RNA水解酶型,如帚曲霉素(α-sarcin),专一水解28SrRNA第4325—4326位之间的磷酸二酯键;(2)RNA N-糖苷酶(RNA N-glycosidase)型,如蓖麻蛋白A     

核糖体RNA拓扑学与RNA N－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.RNA N-糖苷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.Ricin A链是研究得最早和最详细的RNA N-糖苷酶,迄今已发现有二十五种核糖体失活蛋白具有RNA N-糖苷酶活性.RNA N-糖苷酶作用于28S rRNA的α-sarcin结构域,改变核糖体的构象而使其失活.     

利用外源RNA作为内参结合qPCR准确检测SF2蛋白RIP富集RNA的效率

利用实时荧光定量PCR技术(Real-time Quantitative polymerase chain reaction,q PCR)检测0.1%甲醛交联RNA免疫共沉淀(RNA Immunoprecipitation,RIP)富集SF2蛋白相互作用的RNA效率,为研究可变剪接因子SF2相互作用RNA的富集效率提供准确的质检方案。采用RIP技术获取He La细胞中SF2蛋白相互作用的RNA,等比例加入外源酿酒酵母(BY4741)RNA,并以其β-actin作为内参基因,利用q PCR检测RIP富集SF2蛋白相互作用RNA的效率。结果显示,0.1%甲醛交联RIP技术特异性捕获得到SF2蛋白-RNA复合物;q PCR技术检测阳性基因PABP、Srsf1在SF2抗体捕获的产物和Ig G抗体的产物中相应RNA的浓度差异均在60倍以上。利用q PCR技术,以外源酿酒酵母(BY4741)RNA作为内参,能快速、准确定量检测RNA的富集效率。     

Simultaneous existence of cinnamomin (a type II RIP) and small amount of its free A- and B-chain in mature seeds of camphor tree

Cinnamomin, a type II ribosome-inactivating protein (RIP), was isolated from the mature seeds of camphor tree (Cinnamomum camphora). In this paper, small amount of free A- and B-chain of cinnamomin were found to be present in the mature seed cell of C. camphora besides the intact cinnamomin. Our results demonstrated that camphorin, a type I RIP previously reported to coexist with cinnamomin in the seeds of C. camphora, actually was the A-chain of cinnamomin. The percentage of free A- and B-chain in the total cinnamomin was 2.6-2.8% in the seed extract. Of these free A- and B-chain approximate 80% already existed in the seed cell, only about 20% were produced during the purification operation. As the enzymatic activity to reduce disulfide bond of cinnamomin in the seed extract of C. camphora was detected, we proposed that the free A- and B-chain were derived from the enzymatic reduction of the interchain disulfide bond of cinnamomin. It was demonstrated that the endogenous type II RIPs of several plant species, such as Cinnamomum porrectum, Cinnamomum bodinieri and Ricinus communis, could be enzymatically reduced into the free A- and B-chain in their respective seed cells. The function of the free A-chain in the seed cell and the possibility that metabolic enzymes might be involved in the reduction of the interchain disulfide bond of type II RIPs in vivo are discussed.     

Cinnamomin: a multifunctional type II ribosome-inactivating protein

Plant ribosome-inactivating proteins (RIPs) are a group of toxic proteins that can irreversibly inactivate ribosomes by specifically removing the conserved adenine base from the "Sarcin/Ricin domain" of the 28S RNA in ribosome. Cinnamomin is a novel type II RIP isolated in our laboratory from the mature seeds of camphor tree. Besides site-specific deadenylation of the A4324 in the Sarcin/Ricin domain of rat ribosome, this protein could also release the adenine base from DNA molecules at multiple sites and from AMP, ADP, dAMP and adenosine. Furthermore, cinnamomin displays cytotoxicity to carcinoma cells and insect larvae by modifying their ribosomal RNA. These functions possessed by cinnamomin shed a new light on the possible application of cinnamomin in the field of immunotoxin design and transgenic reagents. In this review, we introduce the major recent results on cinnamomin obtained in our laboratory, including purification of this protein, characterization of its enzymatic mechanism, structure and function, gene pattern, physiological role and its biological implications in cytotoxicity.     

樟树核糖体失活蛋白在种子成熟过程中的动态变化与特性

樟树种子中存在着ｃｉｎｎａｍｏｍｉｎ与ｃａｍｐｈｏｒｉｎ两种新的核糖体失活蛋白,电泳分析与Ｗｅｓｔｅｒｎ杂交结果表明ｃｉｎｎａｍｏｍｉｎ在９、１０、１１月份种子中的含量分别是８．９％,２６．８％和１１．５％,以１０月份种子的含量为最高。ｃａｍｐｈｏｒｉｎ的含量则分别为１．７％,２．５％与４．６％,随着种子的成熟而不断增加。８月份的幼嫩种子中检测不出ｃｉｎｎｍａｍｏｍｉｎ与ｃａｍｐｈｏｒｉｎ．这表明樟树核糖体失活蛋白的表达受到了发育进程的时态调控．樟树叶片中可能不存在ｃｉｎｎａｍｏｍｉｎ与ｃａｍｐｈｏｒｉｎ,即两者的合成似乎具有一定的组织特异性．ｃｉｎｎａｍｏｍｉｎ与ｃａｍｐｈｏｒｉｎ均为糖蛋白。     

The lower cytotoxicity of cinnamomin (a type II RIP) is due to its B-chain

The cytotoxicity of intact cinnamomin (a type II ribosome-inactivating protein, RIP) and the RNA N-glycosidase activity of cinnamomin A-chain have been studied and compared with those of ricin. Cinnamomin A-chain exhibits a similar RNA N-glycosidase activity in inhibiting in vitro protein synthesis compared with that of ricin, whereas the cytotoxicity to BA/F3beta cells of intact cinnamomin is markedly lower than intact ricin. In order to demonstrate that it is the B-chains of the two RIPs that bear the difference in cytotoxicity, two hybrid RIPs are prepared from the purified A-/B-chains of cinnamomin and ricin by the disulfide exchange reaction. It has been found that hybrid RIP constructed from cinnamomin A-chain and ricin B-chain is more toxic to BA/F3beta cells than the native cinnamomin, and equivalent to the native ricin. However, the cytotoxicity to BA/F3beta cells of the hybrid RIP constructed from the ricin A-chain and cinnamomin B-chain is lower than ricin, equivalent to the native cinnamomin. Furthermore, the bound amounts of two B-chains on the cell surface are determined by the method of direct cellular ELISA and Scatchard analysis of the binding of the two B-chains indicates that cinnamomin and ricin share similar binding sites with different affinity.     

Studies of three genes encoding Cinnamomin (a type II RIP) isolated from the seeds of camphor tree and their expression patterns

Cinnamomin, which has three isoforms, is a type II ribosome-inactivating protein (RIP) purified from the mature seeds of camphor tree (Cinnamomum camphora). In a previous study, an incomplete cDNA that encoded the A- and B-chain of Cinnamomin but lacked signal peptide sequence was cloned. In the present paper, its full-length cDNA was obtained by 5' rapid amplification of cDNA ends (5'RACE). Subsequently, polymerase chain reaction (PCR) amplification of its genomic DNA was performed. Unexpectedly, sequence analysis of the PCR products revealed three cinnamomin genes with >98.0% sequence identity. One of them corresponded to the published cDNA and was designated as cinnamomin I, whereas the other two genes were named as cinnamomin II and cinnamomin III, respectively. RT-PCR amplification of the cDNAs of cinnamomin II and III manifested that these two genes were functional. The three genes have no intron. Three Cinnamomin precursors that were inferred from the cDNA sequence of three cinnamomin genes exhibited relatively high sequence homology with other type II RIPs. Northern blot analysis demonstrated that the cinnamomin genes only expressed in cotyledons of C. camphora seeds and the acmes of expression emerged at 75-90 DAF when seeds were close to maturity. It is proposed that the three cinnamomin genes may encode three isoforms of Cinnamomin. The physiological function of Cinnamomin in C. camphora seeds is briefly discussed.     

Ribosome-inactivating proteins in edible plants and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata

The basic protein fraction of tissue extracts from 40 edible plants inhibited cell-free protein synthesis and released adenine from herring sperm DNA, thus having adenine glycosylase activity. This suggested the presence of ribosome-inactivating proteins (RIPs) in the plant extracts. This indication was further strengthened by the presence of the two activities after a partial chromatographic purification of three extracts, including that from Lycopersicon esculentum (tomato), which had very low activity. From the extract of Cucurbita moschata (pumpkin), the most active one, a glycoprotein of 30,665 Da was purified which had the properties of a RIP, in that (i) it inhibited protein synthesis by a rabbit reticulocyte lysate with IC50 (concentration giving 50% inhibition) 0.035 nM (1.08 ng ml(-1)) and by HeLa, HT29 and JM cells with IC50 in the 100 nM range, (ii) deadenylated hsDNA and other polynucleotidic substrates, and (iii) depurinated yeast rRNA at a concentration of 0.1 ng ml(-1), all values being comparable to those of other RIPs. The C. moschata RIP gave a weak cross-reaction only with an antiserum against dianthin 32, but not with antisera against other RIPs, and had superoxide dismutase, antifungal and antibacterial activities.     

Studies on the anti-mitogenic, anti-phage and hypotensive effects of several ribosome inactivating proteins

An investigation was conducted to compare the anti-mitogenic, anti-phage and hypotensive activities of several ribosome inactivating proteins (RIPs) in order to ascertain whether the RIPs differed in their potencies in the various bioassays. Agrostin, luffin and saporin elicited a dose-dependent suppression of the mitogenic response of murine splenocytes to concanavalin A. The three RIPs were approximately equipotent in this regard, with near maximal inhibition attained at a dose of 83 nM and approximately 50% inhibition at 830 pM. Trichosanthin was slightly more potent than the three aforementioned RIPs. All of these RIPs were capable of inhibiting the replication of phage M13 in the bacterium Escherichia coli, the ranking of potencies being luffin>trichosanthin>agrostin when tested at a concentration of 3.5 microM. The RIPs gelonin and saporin did not exert a conspicuous antiviral effect at the same dose. After intravenous administration into normotensive rats via the external jugular vein, the RIPs saporin, trichosanthin, gelonin and momordin evoked a mild hypotensive response while luffin and agrostin were inactive. The hypotensive response, however, lacked dose dependence. The RIPs trichosanthin, momordin and gelonin did not affect the blood pressure response to angiotensin I. Chemical modification of the arginine residues of the RIPs brought about a reduction in their ability to inhibit cell-free translation. It appears that the ranking of potency of RIPs in one bioassay was different from the rankings in other assays.     

Cinnamomin a Versatile Type Ⅱ Ribosome-inactivating Protein

Ribosome-inactivating proteins (RIPs) are a groupof ribotoxins widely distributed in the plant kingdomas well as in certain fungi, algae and bacteria. RIPs havebeen thoroughly reviewed in references [1–6]. Theseproteins act as RNA N-glycosidase (rRNA N-glycosidase,EC 3.2.2.22) to specifically remove an adenine fromthe universally conserved sarcin/ricin domain (S/R domain)of the largest RNA in ribosome [7–9] and to render itincapable of carrying out protein synthesis (Fig. 1). Based …     

Ribosome-inactivating proteins in edible plants and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata

The basic protein fraction of tissue extracts from 40 edible plants inhibited cell-free protein synthesis and released adenine from herring sperm DNA, thus having adenine glycosylase activity. This suggested the presence of ribosome-inactivating proteins (RIPs) in the plant extracts. This indication was further strengthened by the presence of the two activities after a partial chromatographic purification of three extracts, including that from Lycopersicon esculentum (tomato), which had very low activity. From the extract of Cucurbita moschata (pumpkin), the most active one, a glycoprotein of 30,665 Da was purified which had the properties of a RIP, in that (i) it inhibited protein synthesis by a rabbit reticulocyte lysate with IC₅₀ (concentration giving 50% inhibition) 0.035 nM (1.08 ng ml⁻¹) and by HeLa, HT29 and JM cells with IC₅₀ in the 100 nM range, (ii) deadenylated hsDNA and other polynucleotidic substrates, and (iii) depurinated yeast rRNA at a concentration of 0.1 ng ml⁻¹, all values being comparable to those of other RIPs. The C. moschata RIP gave a weak cross-reaction only with an antiserum against dianthin 32, but not with antisera against other RIPs, and had superoxide dismutase, antifungal and antibacterial activities.     

Cytotoxicity of two new ribosome-inactivating proteins,cinnamomin and camphorin,to carcinoma cells

Cinnamomin (two-chain) and camphorin (single-chain), two novel ribosome-inactivating proteins (RIPs) purified from the seeds of Cinnamomum camphora, produced inhibitory effects in cultured carcinoma cells. The IC₅₀ of cinnamomin to the human hepatocarcinoma cell-line 7721 and the melanoma cell-line M21 were 18·8 nmol and 11·7 nmol respectively. The IC₅₀ of camphorin to the human hepatocarcinoma cell-line 7721 was 59 nmol, whereas the melanoma cell-line M21 was not susceptible to camphorin. Furthermore, cinnamomin exhibited a remarkable inhibitory effect on the growth of solid melanoma in the skin of the nude mouse. An R-fragment could be isolated from ribosomes of cinnamomin- or camphorin-treated carcinoma cells after incubation with acidic aniline, indicating that the cytotoxicity of these two new RIPs to carcinoma cells might result from modification to the ribosomes.