核糖体失活蛋白抗HIV-1作用的研究进展

核糖体失活蛋白(RIPs)抗HIV-1活性研究已有十几年的历史。RIPs类化合物代表了抗HIV/AIDS天然产物或先导化合物发展的一个重要方向。本文从介绍RIPs的酶活性及其抗HIV-1活性入手，对RIPs抗HIV-1的可能机制，从与RIPs酶活性的关系、诱导HIV-1感染细胞的凋亡及相应的信号转导、诱发活性氧的产生，以及对HIV-1整合酶的抑制作用等几个方面做了较详尽的阐述，并对RIPs的结构修饰和抗HIV-1构效关系进行了综述。对RIPs类化合物在抗病毒领域进行深入而系统地研究，能拓宽其在抗HIV/AIDS临床上的进一步应用。     

核糖体失活蛋白研究进展

核糖体失活蛋白是一类毒蛋白,主要存在于植物当中,在真菌和细菌中也有发现.其共同特点是具有N-糖苷酶活性,能水解生物核糖体大亚基rRNA颈环结构上特定位点的腺嘌呤,使核糖体失活,从而抑制了蛋白质合成.本文对核糖体失活蛋白的主要性质、应用以及国内外有关这类蛋白的研究进展加以概述.     

核糖体失活蛋白研究进展

核糖体失活蛋白是一类毒蛋白, 主要存在于植物当中, 在真菌和细菌中也有发现。其共同特点是具有N-糖苷酶活性, 能水解生物核糖体大亚基rRNA颈环结构上特定位点的腺嘌呤, 使核糖体失活, 从而抑制了蛋白质合成。本文对核糖体失活蛋白的主要性质、应用以及国内外有关这类蛋白的研究进展加以概述。     

植物的核糖体失活蛋白及其应用

对植物的核糖体失活蛋白的分类、性质、功能和应用的研究进展作概述。     

苦瓜的核糖体失活蛋白

核糖体失活蛋白是一类专一修饰核糖体的大亚基rRNA从而抑制蛋白质生物合成的蛋白毒素,可分为Ⅰ-型和Ⅱ-型两种类型。苦瓜中含有多种Ⅰ-型核糖体失活蛋白,如α-苦瓜素,β-苦瓜素和MAP30等,这些蛋白成分具有抗肿瘤、抗病毒和抗艾滋病等功能,因而近年来引起人们广泛的关注。对苦瓜核糖体失活蛋白的研究进展和应用前景进行了综述。     

植物核糖体失活蛋白及其应用进展

植物核糖体失活蛋白（ribosome-inactivating proteins,RIPs）是一类作用于真核细胞rRNA,并破坏其核糖体结构,抑制蛋白质生物合成的毒蛋白,主要应用在农业和医学领域。在农业领域,主要应用在转基因植物中,增强其抗病毒、抗菌以及抗虫活性。在医学领域,主用应用于抗肿瘤、抗艾滋病病毒等研究中。对核糖体失活蛋白的一些性质和应用进展进行综述。     

核糖体失活蛋白及核糖体拓扑结构的研究进展（续完）

核糖体失活蛋白及核糖体拓扑结构的研究进展（续完）李向东刘望夷（中国科学院上海生物化学研究所,上海２０００３１）关键词核糖体失活蛋白核糖体拓扑结构ＲＮＡＮ－糖苷酶２．核糖体拓扑结构的研究核糖体是由数十种生物大分子（ＲＮＡ和蛋白质）构成的。早期普遍接受的...     

单链核糖体失活蛋白的核糖核酸酶活性

以芹菜4.5SRNA为底物, 在pH5.0的条件下, 5种纯核糖体失活蛋白:天花粉蛋白、苦瓜子蛋白、肥皂草蛋白、丝瓜素毒蛋白和多花白树毒蛋白均显示出核糖核酸酶活性, 放射自显影图显示出它们对RNA分子中的各种碱基具有不同的敏感性.     

核糖体失活蛋白及核糖体拓扑结构的研究进展

天花粉毒蛋白使核糖体失活的分子机制是它有ＲＮＡＮ－糖苷酶的作用。从樟树种子中纯化的两种新的核糖体失活蛋白（ＲＩＰ）——辛纳毒蛋白和克木毒蛋白也都具有ＲＮＡＮ－糖苷酶和依赖超螺旋结构的核酸内切酶活性。辛纳毒蛋白还有杀虫活性;克木毒蛋白还有超氧化物歧化酶活性。被ＲＮＡＮ－糖苷酶失活的核糖体用硼氢化钠还原或氨基酸加成反应可部分地复活,这表明失活的核糖体ＲＮＡ上产生的一个活泼醛基对其失活起着重要作用。工作中建立了荧光标记在凝胶上测定小分子ＲＮＡ序列和定性测定糖蛋白的两种新方法。     

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。     

Anti-HIV-1 property of trichosanthin correlates with its ribosome inactivating activity

Trichosanthin (TCS) is a type I ribosome inactivating (RI) protein possessing anti-tumor and antiviral activity, including human immunodeficiency virus (HIV). The mechanism of these actions is not entirely clear, but is generally attributed to its RI property. In order to study the relationship between the anti-HIV-1 activity of TCS and its RI activity, three TCS mutants with different RI activities were constructed by using site-directed mutagenesis. The anti-HIV-1 activities of the three mutants were tested in vitro. Results showed that two TCS mutants, namely TCS(M(120-123)), TCS(E160A/E189A), with the greatest decrease in RI activity, lost almost all of the anti-HIV activity and cytopathic effect. Another mutant TCS(R122G), which exhibited a 160-fold decrease in RI activity, retained some anti-HIV activity. The results from this study suggested that RI activity of TCS may have significant contribution to its anti-HIV-1 property.     

Ribosome Inactivating Proteins from Plants Inhibiting Viruses

Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins, isolated from plants, are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV). Most of the research work related to RIPs has been focused on antiviral activity agains...     

Site-directed PEGylation of trichosanthin retained its anti-HIV activity with reduced potency in vitro

Trichosanthin (TCS) was the first ribosome inactivating protein found to possess anti-HIV-1 activity. Phase I/II clinical trial of this compound had been done. Antigenicity and short plasma half-life were the major side effects preventing further clinical trial. Modification of TCS is therefore necessary to revive the interest to develop this compound as an anti-HIV agent. Three potential antigenic sites (Ser-7, Lys-173, and Gln-219) were identified by computer modeling. Through site-directed mutagenesis, these three antigenic amino acids were mutated to a cysteine residue resulting in 3 TCS mutants, namely S7C, K173C, and Q219C. These mutants were further coupled to polyethylene glycol with a molecular size of 20 kDa (PEG) via the cysteine residue. This produced another three TCS derivatives, namely PEG_20k-S7C, PEG_20k-K173C, and PEG_20k-Q219C. PEGylation had been widely used recently to decrease immunogenicity by masking the antigenic sites and prolong plasma half-life by expanding the molecular size. The in vitro anti-HIV-1 activity of these mutants and derivatives was tested. Results showed that the anti-HIV-1 activity of S7C, K173C, and Q219C was decreased by about 1.5- to 5.5-fold with slightly lower cytotoxicity. On the other hand, PEGylation produced larger decrease (20- to 30-fold) in anti-HIV activity. Cytotoxicity was, however, weakened only slightly by about 3-fold. The in vitro study showed that the anti-HIV activity of PEGylated TCS was retained with reduced potency. The in vivo activity is expected to have only slightly changed due to other beneficial effects like prolonged half-life.     

Independency of anti-HIV-1 activity from ribosome-inactivating activity of trichosanthin

Trichosanthin (TCS) is a type I ribosome-inactivating (RI) protein possessing multiple biological and pharmacological activities. Its major action is inhibition of human immunodeficiency virus (HIV) replication but the mechanism is still elusive. All evidences showed that this action is related to its RI activity. Previous studies found that TCS mutants with reduced RI activity simultaneously lost some anti-HIV activity. In this study, an exception was demonstrated by two TCS mutants retaining almost all RI activity but were devoid of anti-HIV-1 activity. Five mutants were constructed by using site-directed mutagenesis with either deletion or addition of amino acids to the C-terminal sequence. Results showed that the RI activity of mutants with C-terminal deletion mutants (TCS(C2), TCS(C4), and TCS(C14)) decreased by 1.2-3.3-fold with parallel downshifting of its anti-HIV-1 activity (1.4-4.8-fold). Another two mutants, TCS(C19aa) and TCS(KDEL) having 19 amino acid extension and a KDEL signal sequence added to the C-terminal sequence, retained all RI activity but subsequently lost most of the anti-HIV-1 activity. These findings suggested that ribosome inactivation alone might not be adequate to explain the anti-HIV action of TCS.     

重组蓖麻毒素A链与几种天然单链核糖体失活蛋白的活性研究

采用ｐＥｘＳｅｃⅠ载体系统进行了蓖麻毒素Ａ链的原核表达,经ＣＭ－Ｓｅｐｈａｒｏｓｅ一步纯化后,获得了纯度约８０％的重组蓖麻毒素Ａ链．将其与几种天然单链核糖体失活蛋白进行了超螺旋ＤＮＡ裂解研究和无细胞体系中蛋白合成抑制试验,结果表明,重组蓖麻毒素Ａ链具有类似于天然单链核糖体失活蛋白的活性,但两种测活方法之间没有明显的相关性     

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。     

黑木耳菌丝体核糖体失活蛋白的研究

目的：由悬浮培养的黑木耳菌丝体中分离纯化黑木耳的核糖体失活蛋白,对其生化性质及生物学活性进行研究。方法：实验中采用了DEAE-离子交换纤维素,Affi-Gel Blue Gel亲和与Bio-Gel 100柱层析方法。结果：从100g悬浮培养黑木耳菌丝体中得到4.14mg的核糖体失活蛋白,命名为Auriculin。同时证明它在家兔网织红细胞裂解系统中具有抑制蛋白质的翻译活性。结论：经试验证明和文献检索,Auriculin为黑木耳菌分离纯化获得的核糖体失活蛋白,一种新蛋白质。     

Isolation and Characterization of cDNAs Encoding Ribosome Inactivating Protein from Dianthus sinensis L.

To isolate a ribosome inactivating protein (RIP) gene, six plant species were surveyed for antiviral activity. Crude proteins extracted from these plants were tested for the antiviral activity against tobacco mosaic virus (TMV) in Nicotiana glutinosa. All the plants, Spinacia oleracea, Amaranthus lividus, Dianthus superbus, Dianthus sinensis and Celosia cristata, with an exception of Oenanthe stolonifera, presented 70–90% inhibition of viral infectivity. In an attempt to search for the RIP gene from D. sinensis, partial cDNA was obtained by polymerase chain reaction (PCR) of the poly(A)⁺ RNA from D. sinensis leaves. DNA gel blot analysis showed that D. sinensis has multi-copy RIP genes. The expression of RIP gene was investigated in the flower, leaf, root and stem of D. sinensis, and was found to be most abundant in the leaf. Using the partial cDNA as a probe, seven full-length cDNAs were isolated from a library prepared from D. sinensis leaves. They were divided into three groups on the basis of their nucleotide sequence homology. The three representative clones, cDsRIP1, cDsRIP2 and cDsRIP3 were completely sequenced. They all had an open reading frame of 882 bp. The cDsRIP2 showed 79% homology with dianthin 30 and saporin genes; 59% with PAP and Mirabilis antiviral protein MAP genes. From the analysis of deduced amino acid sequences, it was predicted that D. sinensis RIP cDNAs might have a putative signal peptide of 23 amino acid residues at their N-terminus. When the cDNA was expressed in E. coli, the bacteria was unable to grow upon IPTG induction, suggesting that expression of the gene renders toxicity to E. coli cells.     

Purification and Characterization of a New Ribosome Inactivating Protein from Cinchonaglycoside C-treated Tobacco Leaves

A new ribosome-inactivating protein(RIP)with a molecular weight of 31 kDa induced by Cinchonaglycoside C(1)designatedCLP31,was isolated from tobacco leaves.Analysis of this protein sequence indicated that it belongs to the RIP family and itwas distinct from the other plant RIPs reported previously at its N-terminal amino acid sequence.CIP31 can directly impairsynthesis of coat protein(CP)of tobacco mosaic virus(TMV),which resulted in inhibition of TMV long distance movementand multiplication in tobacco plants at concentrations of ng/mL.Furthermore,no toxicity was shown to the growth andfertility of the plants.CIP31 was synthesized only in the presence of Cinchonaglycoside C(1)and was independent of thesalicylic acid(SA)signal pathway.We provided evidence for the SA-independent biological induction of resistance.     

Purification and Characterization of a New Ribosome Inactivating Protein from Cinchonaglycoside C-treated Tobacco Leaves

A new ribosome-inactivating protein （RIP） with a molecular weight of 31 kDa induced by Cinchonaglycoside C （1） designated CIP31, was isolated from tobacco leaves. Analysis of this protein sequence indicated that it belongs to the RIP family and it was distinct from the other plant RIPs reported previously at its N-terminal amino acid sequence. CIP31 can directly impair synthesis of coat protein （CP） of tobacco mosaic virus （TMV）, which resulted in inhibition of TMV long distance movement and multiplication in tobacco plants at concentrations of ng/mL. Furthermore, no toxicity was shown to the growth and fertility of the plants. CIP31 was synthesized only in the presence of Cinchonaglycoside C （1） and was independent of the salicylic acid （SA） signal pathway. We provided evidence for the SA-independent biological induction of resistance.