人核糖核酸酶A家族生物学功能的结构基础

人核糖核酸酶A(ribonuclease A, RNaseA)家族成员有13个，分别为RNase1-RNase13,它们具有很高的序列相似性，大多含有6～8个半胱氨酸并形成分子内二硫键，以维持特有的空间结构。其中，RNase1-RNase8具有多种生物活性，可概括为3类：涉及核糖核酸转录后的剪切、修饰和降解；具有抗细菌、抗真菌和抗病毒活性；以及机体免疫调节作用。而RNase9-RNase13不具有核糖核酸酶活性。因此，本文将重点对RNaseA家族成员RNase1-RNase8的结构与功能研究进行综述，重点概述决定RNaseA生物学功能的结构特征，以期指导以RNaseA为基础的抗微生物药物开发及RNaseA在机体免疫中的功能研究。     

细胞毒性RNase及其抗肿瘤活性

核糖核酸酶(ribonuclease,RNase)是一类核酸水解酶,它们广泛存在于动植物中,除了具有水解RNA的活性外,有的还有一定的细胞毒性。根据结构的相似性,这些RNase属于RNase A超家族(RNase A superfamily)。RNaseA超家族包含了以牛胰核糖核酸酶为原型的不同来源的脊椎动物核糖核酸酶。细胞毒性RNase显示出抑制肿瘤细胞生长的活性,因而有望应用于肿瘤治疗中。本文对RNase A超家族中的几个成员棗核糖核酸酶A(ribonuclease A,RNaseA)、豹蛙抗癌酶(onconase,ONC)、牛蛙核糖核酸酶(Rana catesbeiana ribonuclease,RC-RNase)、牛精液核糖核酸酶(bovine seminal ribonuclease,BS-RNase)和amphinase(Amph)的结构及抗肿瘤活性进行了综述。     

棉花两个β-甘露糖苷酶cDNA的克隆及其特征

从陆地棉纤维cDNA文库中分离出两个B-甘露糖苷酶的cDNA克隆,GhManAl和GhManA2.它们的开放读码框编码长度分别为834个氨基酸和976个氨基酸的多肽序列,这两个多肽C-末端的747个氨基酸残基是完全一致的,而N-末端的序列差异较大.GhManAl和GhManA2均属于糖基水解酶家族2的成员,它们与其它植物来源的该家族中β-甘露糖苷酶之间具有较高的同源性,而与非植物来源的β-甘露糖苷酶之间的同源性较低,但不同蛋白序列中均存在糖基水解酶家族2的酶催化活性所需的两个Glu保守残基.这两个多肽的N-末端均没有信号肽序列,因此可能为胞内酶.从表达特征来看,GhManA1属于组成型表达基因,而GhManA2则为纤维细胞优势表达基因.     

RNase A超家族多样性的产生及宿主防御

胰核糖核酸酶家族也被称为RNase A家族,包含了与牛胰核糖核酸酶同源的所有脊椎核糖核酸酶.从20世纪初.RNase A超家族就成为生物化学、结构生物学、酶学、进化学等领域的研究热点.最新的进化研究显示,脊椎动物RNase A家族起源于宿主防御功能,本文就RNaseA超家族多样性的产生及其宿主防御功能进行综述.     

核糖核酸酶A家族典型成员在肿瘤发生中的作用

人体中核糖核酸酶A(ribonuclease A,RNaseA)家族包含8个典型成员(RNase 1至RNase 8)。已有研究显示，除RNase 8外，该家族其它典型成员影响了胰腺癌、结直肠癌、膀胱癌、乳腺癌和皮肤癌等多种肿瘤的发生发展。在肿瘤发生过程中，特定RNase表达量及糖基化修饰会发生显著改变，是肿瘤诊断的潜在标志物；它们能以多种机制参与肿瘤发生、生长和转移等过程，有望成为肿瘤治疗的靶点；而部分成员则具有杀伤肿瘤细胞、抑制肿瘤发展的功能，存在临床开发成肿瘤治疗药物的可能。具体而言，RNase 1通过核糖核酸酶活性依赖的细胞毒性和细胞外RNA降解功能，发挥直接杀伤肿瘤细胞或降低局部炎症而抑制肿瘤生长的作用；RNase 1还能结合并激活促红细胞生成素，产生肝细胞癌受体相互作用蛋白A4 (erythropoietin-producing hepatocellular carcinoma receptor-interacting protein A4, EphA4)信号通路，促进乳腺癌的发生。RNase 2和RNase 3是嗜酸性粒细胞颗粒蛋白质的重要成分，依赖于阳离子性及核糖核酸酶...     

决明查尔酮合成酶基因的克隆及序列分析

以决明(Cassia tora)为实验材料,利用RT-PCR和RACE技术,从决明嫩叶中克隆出查尔酮合成酶(Chal-one synthase,CHS)基因,其cDNA全长为1 459 bp,编码一个由390个氨基酸残基组成的多肽.氨基酸序列分析表明,决明CHS基因的氨基酸序列中含有44.61%的中性疏水氨基酸,29.74%的中性亲水氨基酸,12.56%的酸性氨基酸和13.O8%的碱性氨基酸.决明CHS基因的氨基酸序列中具有CHS家族酶系的氨基酸保守残基,包括结合底物CoA的结合残基及催化聚酮合成的催化残基,表明其可能参与聚酮化合物的合成.决明与其它植物CHS的氨基酸序列的进化分析表明,其与同为豆科决明属的翼叶决明(Cassia alata)的同源性较近,并且CHS家族可以分为CHS亚家族与非CHS亚家族.将得到的序列提交GenBank,登录号为EU430077.     

禾谷类作物淀粉合酶

淀粉合酶是禾谷类作物淀粉合成所必需的一类酶.根据淀粉合酶家族成员的氨基酸序列的相似性,分别介绍了一个颗粒性淀粉合酶亚家族和四个可溶淀粉合酶亚家族的组成、基因结构和表达特点,并从转录、转录后和翻译后水平上对这些基因的表达调控做了概述.     

核糖核酸酶A超家族：不仅仅是一组降解RNA的酶

核糖核酸酶A超家族(ribonuclease A superfamily; RNase A superfamily),也称脊椎动物分泌型核糖核酸酶超家族(vertebrate secreted ribonucleases superfamily),是二十世纪蛋白质结构、酶学和分子进化领域研究最多最广泛的核糖核酸酶家族。自上世纪初期从牛胰腺中分离鉴定第一个成员以来，已从哺乳动物、两栖动物、爬行动物、鸟和鱼等几百种动物中鉴定了几千个成员。早期对该家族成员的研究不仅促进了蛋白质化学技术的发展，而且为现代生物学研究奠定了基础。目前已知人的核糖核酸酶A超家族成员包括8个典型成员(RNase 1～RNase 8)和5个非典型成员(RNase 9～RNase 13)。功能方面，曾一度以为该家族成员只具有降解核糖核酸的能力。随着血管生成素(angiogenin; RNase 5)、嗜酸性粒细胞衍生神经毒素(eosinophils-derived neurotoxin, EDN; RNase 2)、嗜酸性粒细胞阳离子蛋白(eosinophils cationic protein, ECP; RNase ...     

Onconase研究与开发的最新进展

Onconase是一种在北方豹蛙(Rana pipiens)卵母细胞和早期胚胎内存在的核糖核酸酶,是RNase A超家族中的一员,研究证实它在体内外对多种肿瘤均具显著杀伤作用。Onconase目前已经作为抗肿瘤药物上市,用于治疗恶性间皮瘤。Onconase结构独特,高度稳定。在临床上,Onconase具有副反应较轻,免疫原性低和不易产生耐药性等优点。因此Onconase的研究在学术上和医学应用上均具重要意义。本文综述了Onconase的结构特点、催化专一性、细胞毒性、体内外抗肿瘤活性及其临床应用的最新进展,并讨论了与其相关的一些重要问题。     

TNF家族成员TALL-1及其研究进展

TALL-1是TNF家族成员中最近发现的一个新型细胞因子,由单核细胞和巨噬细胞产生. 人TALL-1由285个氨基酸残基组成,而鼠TALL-1由309个氨基酸残基构成,两者均为Ⅱ型穿膜蛋白质. 人sTALL-1是长为152个氨基酸残基的胞外区片段,对应于C端134～285位氨基酸残基. 重组人sTALL-1具有刺激B细胞增殖、激活NF-κB和JNK以及抑制肿瘤细胞生长等生物学活性. 另外,TALL-1在转基因鼠中的过量表达可引起严重的B细胞增生以及与狼疮有关的自身免疫性疾病. 因此,TALL-1是一个具有多种生物学活性的细胞调控因子.     

Rapid Diversification of RNase A Superfamily Ribonucleases from the Bullfrog, Rana catesbeiana

We present sequences of five novel RNase A superfamily ribonuclease genes of the bullfrog, Rana catesbeiana. All five genes encode ribonucleases that are similar to Onconase, a cytotoxic ribonuclease isolated from oocytes of R. pipiens. With amino acid sequence data from 14 ribonucleases from three Rana species (R. catesbeiana, R. japonica, and R. pipiens), we have constructed bootstrap-supported phylogenetic trees that reorganize these ribonucleases into five distinct lineages--the pancreatic ribonucleases (RNases 1), the eosinophil-associated ribonucleases (RNases 2, 3, and 6), the ribonucleases 4, the angiogenins (RNases 5) and the Rana ribonucleases--with the Rana ribonucleases no more closely related to the angiogenins than they are to any of the other ribonuclease lineages shown. Further phylogenetic analysis suggests the division of the Rana ribonucleases into two subclusters (A and B), with positive (Darwinian) selection (dN/dS > 1.0) and an elevated rate of radical nonsynonymous substitution (dR) contributing to the rapid diversification of ribonucleases within each cluster. This pattern of evolution-rapid diversification via positive selection among sequences of a multigene cluster-bears striking resemblance to what we have described for the eosinophil-associated ribonuclease genes of the rodent Mus musculus, a finding that may have implications with respect the physiologic function of this unique family of proteins.     

抗肿瘤蛋白Onconase的原核细胞表达及细胞毒性检测

本文主要利用pET22b( )表达载体在大肠杆菌中表达一种在两栖类蛙(Ranapipiens)卵细胞内存在的核糖核酸酶-Onconase。通过包涵体复性、蛋白纯化等程序最终获得与天然蛋白活性相似的重组蛋白,并检测Onconase对源于皮肤T细胞淋巴瘤的Hut-78肿瘤细胞的毒性(IC50=0．51μmol／L),证明Onconase用于抗淋巴瘤的可行性。     

A Novel Cationic Ribonuclease with Antimicrobial Activity from Rana dybowskii

A novel ribonuclease (RNase) A superfamily gene (Rdronc) has been cloned from the frog Rana dybowskii. The deduced amino acid sequence shows that it belongs to the ribonuclease A superfamily, with the highest identity, 73%, to Rana pipiens onconase. Adaptive evolution analysis based on maximum likelihood models of codon substitution has been conducted on 10 members of the Rana RNases of subcluster B. Rapid adaptive evolution and multiple positive selection sites have been detected, which indicates that these genes may be evolving under positive selection pressure. Functional assay demonstrates that the recombinant Rdronc protein possesses antimicrobial activity against Gram-negative Escherichia coli and Pseudomonas aeruginosa and weaker antimicrobial activity against Gram-positive Staphylococcus aureus and yeast Candida albicans. Our findings support the hypothesis that ribonuclease A superfamily members may function in host defense of early-diversified vertebrates.     

Quantitative analysis, using MALDI-TOF mass spectrometry, of the N-terminal hydrolysis and cyclization reactions of the activation process of onconase.

Onconase, a member of the ribonuclease superfamily, is a potent cytotoxic agent that is undergoing phase II/III human clinical trials as an antitumor drug. Native onconase from Rana pipiens and its amphibian homologs have an N-terminal pyroglutamyl residue that is essential for obtaining fully active enzymes with their full potential as cytotoxins. When expressed cytosolically in bacteria, Onconase is isolated with an additional methionyl (Met1) residue and glutaminyl instead of a pyroglutamyl residue at position 1 of the N-terminus and is consequently inactivated. The two reactions necessary for generating the pyroglutamyl residue have been monitored by MALDI-TOF MS. Results show that hydrolysis of Met(-1), catalyzed by Aeromonas aminopeptidase, is optimal at a concentration of >or= 3 m guanidinium-chloride, and at pH 8.0. The intramolecular cyclization of glutaminyl that renders the pyroglutamyl residue is not accelerated by increasing the concentration of denaturing agent or by strong acid or basic conditions. However, temperature clearly accelerates the formation of pyroglutamyl. Taken together, these results have allowed the characterization and optimization of the onconase activation process. This procedure may have more general applicability in optimizing the removal of undesirable N-terminal methionyl residues from recombinant proteins overexpressed in bacteria and providing them with biological and catalytic properties identical to those of the natural enzyme.     

The importance of dynamic effects on the enzyme activity: X-ray structure and molecular dynamics of onconase mutants

Onconase (ONC), a member of the RNase A superfamily extracted from oocytes of Rana pipiens, is an effective cancer killer. It is currently used in treatment of various forms of cancer. ONC antitumor properties depend on its ribonucleolytic activity that is low in comparison with other members of the superfamily. The most damaging side effect from Onconase treatment is renal toxicity, which seems to be caused by the unusual stability of the enzyme. Therefore, mutants with reduced thermal stability and/or increased catalytic activity may have significant implications for human cancer chemotherapy. In this context, we have determined the crystal structures of two Onconase mutants (M23L-ONC and C87S,des103-104-ONC) and performed molecular dynamic simulations of ONC and C87S,des103-104-ONC with the aim of explaining on structural grounds the modifications of the activity and thermal stability of the mutants. The results also provide the molecular bases to explain the lower catalytic activity of Onconase compared with RNase A and the unusually high thermal stability of the amphibian enzyme.     

Cytostatic and Cytotoxic Properties of Amphinase: A Novel Cytotoxic Ribonuclease from Rana pipiens Oocytes

Onconase (Onc), is a novel amphibian cytotoxic ribonuclease with antitumor activity, and is currently in a confirmatory phase III clinical trial for the treatment of malignant mesothelioma. It was recently reported that Rana pipiens oocytes contain still another ribonuclease, named Amphinase (Amph). Amph shows 38 – 40 % amino acid sequence identity with Onc; presents as four variants varying between themselves from 87 to 99 % in amino acid sequence identity and has a molecular mass ~ 13,000. In the present study we describe the effects of Amph on growth of several tumor cell lines. All four variants demonstrated cytostatic and cytotoxic activity against human promyelocytic HL-60-, Jurkat T-cell- and U-937 monocytic leukemia cells. The pattern of Amph activity to certain extent resembled that of Onc. Thus, cell proliferation was suppressed at 0.5 – 10.0 µg/ml (40 – 80 nM) Amph concentration with distinct accumulation of cells in G1 phase of the cell cycle. In addition, the cells were undergoing apoptosis, which manifested by DNA fragmentation (presence of “sub-G1” cells, TUNEL-positivity), caspases and serine proteases activation as well as activation of transglutaminase. The cytotostatic and cytotoxic effects of Amph required its ribonuclease activity: the enzymatically inactive Amph-2 having histidine at the active site alkylated was ineffective. The effectiveness and cell cycle specificity was generally similar for all four Amph variants and at the equimolar concentrations was somewhat more pronounced than that of Onc. The observed cytostatic and cytotoxic activity of Amph against tumor cell lines suggests that similar to Onc this cytotoxic ribonuclease may have antitumor activity and find an application in clinical oncology.     

A nuclear localization sequence endows human pancreatic ribonuclease with cytotoxic activity

Some members of the ribonuclease superfamily, such as Onconase, are cytotoxic to cancer cells. This is not the case for human pancreatic ribonuclease. This lack of cytotoxicity is probably a result of the inhibition exerted by the cytosolic ribonuclease inhibitor once the protein has reached the cytosol. Until now, all cytotoxic human pancreatic ribonuclease variants have been described as being resistant to the inhibitor. Here, we report on the characterization of a cytotoxic variant of human pancreatic ribonuclease which has an Arg triplet introduced onto one of its surface-exposed loops. Despite its sensitivity to the inhibitor, this variant, called PE5, was only 5-15 times less cytotoxic than Onconase. When it was taken up by cells, it was only observed within late compartments of the endocytic pathway, probably because the number of molecules transported to the cytosol was too small to allow their visualization. Nuclear import assays showed that the Arg triplet endows PE5 with a nuclear localization signal. In these experiments, PE5 was efficiently transported to the nucleus where it was initially localized in the nucleolus. Although the Arg introduction modified the net charge of the protein and somehow impaired recognition by the cytosolic inhibitor, control variants, which had the same number of charges or were not recognized by the inhibitor, were not toxic. We concluded that targeting a ribonuclease to the nucleus results in cytotoxicity. This effect is probably due to ribonuclease interference with rRNA processing and ribosome assembly within the nucleolus.     

Purification and cloning of cytotoxic ribonucleases from Rana catesbeiana (bullfrog)

Ribonucleases with antitumor activity are mainly found in the oocytes and embryos of frogs, but the role of these ribonucleases in frog development is not clear. Moreover, most frog ribonuclease genes have not been cloned and characterized. In the present study, a group of ribonucleases were isolated from Rana catesbeiana (bullfrog). These ribonucleases in mature oocytes, namely RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by column chromatographs and detected by zymogram assay and western blotting. Characterization of these purified ribonucleases revealed that they were highly conserved in amino acid sequence and had a pyroglutamate residue at their N-termini, but possessed different specific activities, base specificities and optimal pH values for their activities. These ribonucleases were cytotoxic to cervical carcinoma HeLa cells, but their cytotoxicities were not closely correlated to their enzymatic specific activities. Some other amino acid residues in addition to their catalytic residues were implicated to be involved in the cytotoxicity of the frog ribonucleases to tumor cells. Because the coding regions lack introns, the ribonuclease genes were cloned by PCR using genomic DNA as template. Their DNA sequences and amino acid sequences are homologous to those of mammalian ribonuclease superfamily, ~50 and ~25%, respectively.     

Endowing human pancreatic ribonuclease with toxicity for cancer cells

Onconase is an amphibian protein that is now in Phase III clinical trials as a cancer chemotherapeutic. Human pancreatic ribonuclease (RNase 1) is homologous to Onconase but is not cytotoxic. Here, ERDD RNase 1, which is the L86E/N88R/G89D/R91D variant of RNase 1, is shown to have conformational stability and ribonucleolytic activity similar to that of the wild-type enzyme but > 10(3)-fold less affinity for the endogenous cytosolic ribonuclease inhibitor protein. Most significantly, ERDD RNase 1 is toxic to human leukemia cells. The addition of a non-native disulfide bond to ERDD RNase 1 not only increases the conformational stability of the enzyme but also increases its cytotoxicity such that its IC(50) value is only 8-fold greater than that of Onconase. Thus, only a few amino acid substitutions are necessary to make a human protein toxic to human cancer cells. This finding has significant implications for human cancer chemotherapy.     

Three-state thermal unfolding of onconase

Onconase is a member of the ribonuclease A superfamily currently in phase IIIb clinical trials as a treatment for malign mesothelioma due to its cytotoxic activity selective against tumor-cells. In this work, we have studied the equilibrium thermal unfolding of onconase using a combination of several structural and biophysical techniques. Our results indicate that at least one significantly populated intermediate, which implies the exposure of hydrophobic surface and significant changes in the environment around Trp3, occurs during the equilibrium unfolding process of this protein. The intermediate begins to populate at about 30° below the global unfolding temperature, reaching a maximum population of nearly 60%, 10° below the global unfolding temperature.