Pokeweed antiviral protein region Gly209-Lys225 is critical for RNA N-glycosidase activity of the prokaryotic ribosome

Pokeweed antiviral protein (PAP) isolated from Phytolacca americana is a ribosome-inactivating protein (RIP) that has RNA N-glycosidase (RNG) activity towards both eukaryotic and prokaryotic ribosomes. In contrast, karasurin-A (KRN), a RIP from Trichosanthes kirilowii var. japonica, is active only on eukaryotic ribosomes. Stepwise selection of chimera proteins between PAP and KRN indicated that the C-terminal region of PAP (residues 209–225) was critical for RNG activity toward prokaryotic ribosomes. When the region of PAP (residues 209–225) was replaced with the corresponding region of KRN the PAP chimera protein, like KRN, was active only on eukaryotic ribosomes. Furthermore, insertion of the region of PAP (residues 209–225) into the KRN chimera protein resulted not only in the detectable RNG activity toward prokaryotic ribosome, but also activity toward the eukaryotic ribosomes as well that was seven-fold higher than for the original KRN. In this study, the possibility of genetic manipulation of the activity and substrate specificity of RIPs is demonstrated.     

The C-terminal fragment of the ribosomal P protein complexed to trichosanthin reveals the interaction between the ribosome-inactivating protein and the ribosome

Ribosome-inactivating proteins (RIPs) inhibit protein synthesis by enzymatically depurinating a specific adenine residue at the sarcin-ricin loop of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation centre of the ribosome. Here, we present the 2.2 Å crystal structure of trichosanthin (TCS) complexed to the peptide SDDDMGFGLFD, which corresponds to the conserved C-terminal elongation factor binding domain of the ribosomal P protein. The N-terminal region of this peptide interacts with Lys173, Arg174 and Lys177 in TCS, while the C-terminal region is inserted into a hydrophobic pocket. The interaction with the P protein contributes to the ribosome-inactivating activity of TCS. This 11-mer C-terminal P peptide can be docked with selected important plant and bacterial RIPs, indicating that a similar interaction may also occur with other RIPs.     

Trypanosoma cruzi: high ribosomal resistance to trichosanthin inactivation

Trypanosoma cruzi is the parasite causing Chagas Disease. Several results already published suggest that T. cruzi ribosomes have remarkable differences with their mammalian counterparts. In the present work, we showed that trypanosomatid (T. cruzi and Crithidia fasciculata) ribosomes are highly resistant to inactivation by trichosanthin (TCS), which is active against mammalian ribosomes. Differential resistance is an intrinsic feature of the ribosomal particles, as demonstrated by using assays where the only variable was the ribosomes source. Because we have recently described that TCS interacts with the acidic C-terminal end of mammalian ribosomal P proteins, we assayed the effect of a TCS variant, which is unable to interact with P proteins, on trypanosomatid ribosomes. This mutant showed similar shifting of IC(50) values on rat, T. cruzi and C. fasciculata ribosomes, suggesting that the resistance mechanism might involve other ribosomal components rather than the C-terminal end of P proteins.     

Proteomic analysis of the Trypanosoma cruzi ribosomal proteins

Trypanosoma cruzi is a parasite responsible for Chagas disease. The identification of new targets for chemotherapy is a major challenge for the control of this disease. Several lines of evidences suggest that the translational system in trypanosomatids show important differences compared to other eukaryotes. However, there little is known information about this. We have performed a detailed data mining search for ribosomal protein genes in T. cruzi genome data base combined with mass spectrometry analysis of purified T. cruzi ribosomes. Our results show that T. cruzi ribosomal proteins have ∼50% sequence identity to yeast ones. Nevertheless, some parasite proteins are longer due to the presence of several N- or C-terminal extensions, which are exclusive of trypanosomatids. In particular, L19 and S21 show C-terminal extensions of 168 and 164 amino acids, respectively. In addition, we detected two 60S subunit proteins that had not been previously detected in the T. cruzi total proteome; namely, L22 and L42.     

The ribosomal stalk is required for ribosome binding,depurination of the rRNA and cytotoxicity of ricin A chain in Saccharomyces cerevisiae

Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga‐like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the α‐sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic ribosomes. Ricin depurinates the eukaryotic ribosomes more efficiently than the prokaryotic ribosomes, while PAP can depurinate both types of ribosomes. Accumulating evidence suggests that different docking sites on the ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the α‐sarcin/ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild‐type ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the ΔP1 and ΔP2 mutants in vivo and these mutants were more resistant to the cytotoxicity of RTA than the wild‐type cells. We further show that while RTA, Stx1 and Stx2 have similar requirements for ribosome depurination, PAP has different requirements, providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their ribosome specificity.     

Comparative Analysis of the Ribosomal Componentsof the Hydrogenosome-Containing Protist, Trichomonas vaginalis

The ribosomes of the amitochondriate but hydrogenosome-containing protist lineage, the trichomonads, have previously been reported to be prokaryotic or primitive eukaryotic, based on evidence that they have a 70S sedimentation coefficient and a small number of proteins, similar to prokaryotic ribosomes. In order to determine whether the components of the trichomonad ribosome indeed differ from those of typical eukaryotic ribosomes, the ribosome of a representative trichomonad, Trichomonas vaginalis, was characterized. The sedimentation coefficient of the T. vaginalis ribosome was smaller than that of Saccharomyces cerevisiae and larger than that of Escherichia coli. Based on two-dimensional PAGE analysis, the number of different ribosomal proteins was estimated to be approximately 80. This number is the same as those obtained for typical eukaryotes (approximately 80) but larger than that of E. coli (approximately 55). N-Terminal amino acid sequencing of 18 protein spots and the complete sequences of 4 ribosomal proteins as deduced from their genes revealed these sequences to display typical eukaryotic features. Phylogenetic analyses of the five ribosomal proteins currently available also clearly confirmed that the T. vaginalis sequences are positioned within a eukaryotic clade. Comparison of deduced secondary structure models of the small and large subunit rRNAs of T. vaginalis with those of other eukaryotes revealed that all helices commonly found in typical eukaryotes are present and conserved in T. vaginalis, while variable regions are shortened or lost. These lines of evidence demonstrate that the T. vaginalis ribosome has no prokaryotic or primitive eukaryotic features but is clearly a typical eukaryotic type.     

Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses

Trichosanthin (TCS) is a type I ribosome-inactivating protein that inactivates ribosome by enzymatically depurinating the A⁴³²⁴ at the α-sarcin/ricin loop of 28S rRNA. We have shown in this and previous studies that TCS interacts with human acidic ribosomal proteins P0, P1 and P2, which constitute the lateral stalk of eukaryotic ribosome. Deletion mutagenesis showed that TCS interacts with the C-terminal tail of P2, the sequences of which are conserved in P0, P1 and P2. The P2-binding site on TCS was mapped to the C-terminal domain by chemical shift perturbation experiments. Scanning charge-to-alanine mutagenesis has shown that K173, R174 and K177 in the C-terminal domain of TCS are involved in interacting with the P2, presumably through forming charge–charge interactions to the conserved DDD motif at the C-terminal tail of P2. A triple-alanine variant K173A/R174A/K177A of TCS, which fails to bind P2 and ribosomal stalk in vitro, was found to be 18-fold less active in inhibiting translation in rabbit reticulocyte lysate, suggesting that interaction with P-proteins is required for full activity of TCS. In an analogy to the role of stalk proteins in binding elongation factors, we propose that interaction with acidic ribosomal stalk proteins help TCS to locate its RNA substrate.     

Maize Ribosome-Inactivating Protein Uses Lys158–Lys161 to Interact with Ribosomal Protein P2 and the Strength of Interaction Is Correlated to the Biological Activities

Ribosome-inactivating proteins (RIPs) inactivate prokaryotic or eukaryotic ribosomes by removing a single adenine in the large ribosomal RNA. Here we show maize RIP (MOD), an atypical RIP with an internal inactivation loop, interacts with the ribosomal stalk protein P2 via Lys158–Lys161, which is located in the N-terminal domain and at the base of its internal loop. Due to subtle differences in the structure of maize RIP, hydrophobic interaction with the ‘FGLFD’ motif of P2 is not as evidenced in MOD-P2 interaction. As a result, interaction of P2 with MOD was weaker than those with trichosanthin and shiga toxin A as reflected by the dissociation constants (K_D) of their interaction, which are 1037.50±65.75 µM, 611.70±28.13 µM and 194.84±9.47 µM respectively.Despite MOD and TCS target at the same ribosomal protein P2, MOD was found 48 and 10 folds less potent than trichosanthin in ribosome depurination and cytotoxicity to 293T cells respectively, implicating the strength of interaction between RIPs and ribosomal proteins is important for the biological activity of RIPs. Our work illustrates the flexibility on the docking of RIPs on ribosomal proteins for targeting the sarcin-ricin loop and the importance of protein-protein interaction for ribosome-inactivating activity.     

Chagas disease: a homology model for the three-dimensional structure of the Trypanosoma cruzi ribosomal P0 antigenic protein

Ribosomal P proteins form a “stalk” complex in the large subunit of the ribosomes. In Trypanosoma cruzi, the etiological agent of Chagas disease, the complex is formed by five P protein members: TcP0, TcP1α, TcP1β, TcP2α and TcP2β. The TcP0 protein has 34 kDa, and TcP1 and TcP2 proteins have 10 kDa. The structure of T. cruzi P0 and the stalk complex TcP0–TcP1α–TcP1β–TcP2α–TcP2β have not been solved to date. In this work, we constructed a three-dimensional molecular model for TcP0 using homology modeling as implemented in the MODELLER 9v12 software. The model was constructed using different templates: the X-ray structures of the protein P0 from Pirococcus horikoshii, a segment from the Danio renio Ca⁺²/K⁺ channel and the C-terminal peptide (C13) from T. cruzi ribosomal P2 protein; the Cryo-EM structure of Triticum aestivum P0 protein and the NMR structure of Homo sapiens P1 ribosomal protein. TcP0 has a 200-residue-long N-terminal, which is an α/β globular stable domain, and a flexible C-terminal, 120-residue-long domain. The molecular surface electrostatic potential and hydrophobic surface were calculated. The surface properties are important for the C-terminal's antigenic properties. They are also responsible for P0-specific binding to RNA26S and the binding to the P1–P2 proteins. We explored and identified protein interactions that may be involved in conformational stability. The structure proposed in this work represents a first structural report for the TcP0 protein.     

Engineering a switch-on peptide to ricin A chain for increasing its specificity towards HIV-infected cells

Background

Ricin is a type II ribosome-inactivating protein (RIP) that potently inactivates eukaryotic ribosomes by removing a specific adenine residue at the conserved α-sarcin/ricin loop of 28S ribosomal RNA (rRNA). Here, we try to increase the specificity of the enzymatically active ricin A chain (RTA) towards human immunodeficiency virus type 1 (HIV-1) by adding a loop with HIV protease recognition site to RTA.

Methods

HIV-specific RTA variants were constructed by inserting a peptide with HIV-protease recognition site either internally or at the C-terminal region of wild type RTA. Cleavability of variants by viral protease was tested in vitro and in HIV-infected cells. The production of viral p24 antigen and syncytium in the presence of C-terminal variants was measured to examine the anti-HIV activities of the variants.

Results

C-terminal RTA variants were specifically cleaved by HIV-1 protease both in vitro and in HIV-infected cells. Upon proteolysis, the processed variants showed enhanced antiviral effect with low cytotoxicity towards uninfected cells.

Conclusions

RTA variants with HIV protease recognition sequence engineered at the C-terminus were cleaved and the products mediated specific inhibitory effect towards HIV replication.

General significance

Current cocktail treatment of HIV infection fails to eradicate the virus from patients. Here we illustrate the feasibility of targeting an RIP towards HIV-infected cells by incorporation of HIV protease cleavage sequence. This approach may be generalized to other RIPs and is promising in drug design for combating HIV.     

A ribosomal protein is specifically recognized by saporin, a plant toxin which inhibits protein synthesis.

Many plants express enzymes which specifically remove an adenine residue from the skeleton of the 28 S RNA in the major subunit of the eukaryotic ribosome (ribosome inactivating proteins, RIPs). The site of action of RIPs (A4324 in the rRNA from rat liver) is in a loop structure whose nucleotide sequence all around the target adenine is also conserved in those species which are completely or partially insensitive to RIPs. In this paper we identify a covalent complex between saporin (the RIP extracted from Saponaria officinalis) and ribosomal proteins from yeast (Saccharomyces cerevisiae), by means of chemical crosslinking and immunological or avidin-biotin detection. The main complex (mol. wt. congruent to 60 kDa) is formed only with a protein from the 60 S subunit of yeast ribosomes, and is not detected with ribosomes from E. coli, a resistant species. This observation supports the hypothesis for a molecular recognition mechanism involving one or more ribosomal proteins, which could provide a 'receptor' site for the toxin and favour optimal binding of the target adenine A4324 to the active site of the RIP.     

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A₂₆₆₀, which lies in a sequence that is highly conserved in all species.     

Isolation and characterization of two new N-glycosidase type-1 ribosome-inactivating proteins,unrelated in amino-acid sequence,fromPetrocoptis species

Two new N-glycosidase type-1 ribosome-inactivating proteins (RIPs), denoted petroglaucin 1 and petrograndin, respectively, were isolated from the plantsPetrocoptis glaucifolia (Lag.) Boiss sp.viscosa (Rothm.) Laínz andPetrocoptis grandiflora Rothm. These new RIPs do not share H₂N-terminal amino-acid sequence homology with petroglaucin (now denoted as petroglaucin 2), the only other type-1 RIP to be isolated fromP. glaucifolia (Arias et al. (1992) Planta186, 532–540). Petroglaucin 1 shares amino-acid sequence homology with RIPs from Cucurbitaceae while petroglaucin 2 and petrograndin do so with saporins and dianthin 30 (Caryophyllaceae). The new RIPs strongly inhibited protein synthesis at subnanomolar concentrations in rabbit reticulocyte lysates and other eukaryotic cell-free systems, but they were inactive on bacterial ribosomes.     

Solution structure of human P1?P2 heterodimer provides insights into the role of eukaryotic stalk in recruiting the ribosome-inactivating protein trichosanthin to the ribosome

Lateral ribosomal stalk is responsible for binding and recruiting translation factors during protein synthesis. The eukaryotic stalk consists of one P0 protein with two copies of P1•P2 heterodimers to form a P0(P1•P2)₂ pentameric P-complex. Here, we have solved the structure of full-length P1•P2 by nuclear magnetic resonance spectroscopy. P1 and P2 dimerize via their helical N-terminal domains, whereas the C-terminal tails of P1•P2 are unstructured and can extend up to ∼125 Å away from the dimerization domains. ¹⁵N relaxation study reveals that the C-terminal tails are flexible, having a much faster internal mobility than the N-terminal domains. Replacement of prokaryotic L10(L7/L12)₄/L11 by eukaryotic P0(P1•P2)₂/eL12 rendered Escherichia coli ribosome, which is insensitive to trichosanthin (TCS), susceptible to depurination by TCS and the C-terminal tail was found to be responsible for this depurination. Truncation and insertion studies showed that depurination of hybrid ribosome is dependent on the length of the proline-alanine rich hinge region within the C-terminal tail. All together, we propose a model that recruitment of TCS to the sarcin-ricin loop required the flexible C-terminal tail, and the proline-alanine rich hinge region lengthens this C-terminal tail, allowing the tail to sweep around the ribosome to recruit TCS.     

Ribosomal RNAs are tolerant toward genetic insertions: evolutionary origin of the expansion segments

Ribosomal RNAs (rRNAs), assisted by ribosomal proteins, form the basic structure of the ribosome, and play critical roles in protein synthesis. Compared to prokaryotic ribosomes, eukaryotic ribosomes contain elongated rRNAs with several expansion segments and larger numbers of ribosomal proteins. To investigate architectural evolution and functional capability of rRNAs, we employed a Tn5 transposon system to develop a systematic genetic insertion of an RNA segment 31 nt in length into Escherichia coli rRNAs. From the plasmid library harboring a single rRNA operon containing random insertions, we isolated surviving clones bearing rRNAs with functional insertions that enabled rescue of the E. coli strain (Δ7rrn) in which all chromosomal rRNA operons were depleted. We identified 51 sites with functional insertions, 16 sites in 16S rRNA and 35 sites in 23S rRNA, revealing the architecture of E. coli rRNAs to be substantially flexible. Most of the insertion sites show clear tendency to coincide with the regions of the expansion segments found in eukaryotic rRNAs, implying that eukaryotic rRNAs evolved from prokaryotic rRNAs suffering genetic insertions and selections.     

GPIomics: global analysis of glycosylphosphatidylinositol‐anchored molecules of Trypanosoma cruzi

Glycosylphosphatidylinositol (GPI) anchoring is a common, relevant posttranslational modification of eukaryotic surface proteins. Here, we developed a fast, simple, and highly sensitive (high attomole‐low femtomole range) method that uses liquid chromatography‐tandem mass spectrometry (LC‐MSⁿ) for the first large‐scale analysis of GPI‐anchored molecules (i.e., the GPIome) of a eukaryote, Trypanosoma cruzi, the etiologic agent of Chagas disease. Our genome‐wise prediction analysis revealed that approximately 12% of T. cruzi genes possibly encode GPI‐anchored proteins. By analyzing the GPIome of T. cruzi insect‐dwelling epimastigote stage using LC‐MSⁿ, we identified 90 GPI species, of which 79 were novel. Moreover, we determined that mucins coded by the T. cruzi small mucin‐like gene (TcSMUG S) family are the major GPI‐anchored proteins expressed on the epimastigote cell surface. TcSMUG S mucin mature sequences are short (56–85 amino acids) and highly O‐glycosylated, and contain few proteolytic sites, therefore, less likely susceptible to proteases of the midgut of the insect vector. We propose that our approach could be used for the high throughput GPIomic analysis of other lower and higher eukaryotes.     

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA.

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A₂₆₆₀, which lies in a sequence that is highly conserved in all species.     

Biological and antipathogenic activities of ribosome-inactivating proteins from Phytolacca dioica L.

BackgroundThe species from the genus Phytolacca constitute one of the best sources of ribosome-inactivating proteins (RIPs) that have been used both in the therapy against virus and tumors and in the construction of transgenic plants resistant to virus, bacteria, fungi and insects. Here we investigate new activities of three representative RIPs from Phytolacca dioica (dioicin 2, PD-S2 and PD-L4).ResultsThe three RIPs displayed, in addition to already reported activities, rRNA N-glycosylase activities against plant, bacterial and fungal ribosomes. Additionally dioicin 2 and PD-L4 displayed endonuclease activity on a supercoiled plasmid DNA, and dioicin 2 and PD-S2 arrested the growth of the fungus Penicillium digitatum. Furthermore, dioicin 2 induced caspase activation and apoptosis in cell cultures.ConclusionsThe different activities of the RIPs from Phytolacca dioica may explain the antipathogenic properties attributed to these RIPs in plants and their antiviral and antitumoral effects. In spite of the similarity in their rRNA N-glycosylase and DNA polynucleotide:adenosine glycosylase activities, they differed in their activities against viral RNA, plasmid DNA, fungi and animal cultured cells. This suggests that the presence of isoforms might optimize the response of the plant against several types of pathogens.General significanceRIPs from Phytolacca can induce plant resistance or tumor cell death not only by means of ribosome inactivation but also by the activities found in this report. Furthermore, the induction of cell death by different mechanisms turns these RIPs into more useful tools for cancer treatment rendering the selection of RIP-resistant mutants impossible.     

研究: 核糖体失活蛋白及其在黄瓜中的表达分析

核糖体失活蛋白是一类具有高度特异性rRNA N-糖苷酶活性的蛋白,它们能够使原核或真核细胞的核糖体失活因而具有细胞毒性．由于其独特的生物学性质,核糖体失活蛋白被认为在农业和医学中都有着巨大的应用潜力．我们之前的研究表明,黄瓜的基因组中共包含2个2类核糖体失活蛋白基因,分别命名为CumsaAB1和CumsaAB2．以蓖麻毒蛋白Ricin为代表,2类核糖体失活蛋白通常由2条二硫键连接的肽链组成：具有N-糖苷酶活性的A链与具有凝集素活性的B链．本文研究了黄瓜中核糖体失活蛋白的表达情况．亚细胞定位研究表明CumsaAB1经过蛋白分泌通路表达于细胞外,这与蛋白质序列分析显示的CumsaAB1包含一个信号肽而不含转膜区域相一致．对黄瓜的不同生长阶段的不同组织中的转录水平分析表明,CumsaAB1在大部分组织中以极低的水平表达,而CumsaAB2表达水平则明显更高,尤其在第一片真叶阶段和刚开花的植物中．最后,我们使用分子模拟对黄瓜中核糖体失活蛋白的结构及糖结合位点进行了分析．本研究对黄瓜中核糖体失活蛋白的亚细胞定位、表达水平和可能的蛋白质结构进行了研究,为其进一步的生物学功能研究提供了重要信息．