Phosphorylation of the conserved C-terminal domain of ribosomal P-proteins impairs the mode of interaction with plant toxins.

The ribosome is subjected to post-translational modifications, including phosphorylation, that affect its biological activity. Among ribosomal elements, the P-proteins undergo phosphorylation within the C terminus, the element which interacts with trGTPases or ribosome-inactivating proteins (RIPs); however, the role of phosphorylation has never been elucidated. Here, we probed the function of phosphorylation on the interaction of P-proteins with RIPs using the ribosomal P1-P2 dimer. We determined the kinetic parameters of the interaction with the toxins using biolayer interferometry and microscale thermophoresis. The results present the first mechanistic insight into the function of P-protein phosphorylation, showing that introduction of a negative charge into the C terminus of P1-P2 proteins promotes α-helix formation and decreases the affinity of the P-proteins for the RIPs.

Structural basis for the interaction of Shiga toxin 2a with a C-terminal peptide of ribosomal P stalk proteins.

The principal virulence factor of human pathogenic enterohemorrhagic Escherichia coli is Shiga toxin (Stx). Shiga toxin 2a (Stx2a) is the subtype most commonly associated with severe disease outcomes such as hemorrhagic colitis and hemolytic uremic syndrome. The catalytic A1 subunit (Stx2A1) binds to the conserved elongation factor binding C-terminal domain (CTD) of ribosomal P stalk proteins to inhibit translation. Stx2a holotoxin also binds to the CTD of P stalk proteins because the ribosome-binding site is exposed. We show here that Stx2a binds to an 11-mer peptide (P11) mimicking the CTD of P stalk proteins with low micromolar affinity. We cocrystallized Stx2a with P11 and defined their interactions by X-ray crystallography. We found that the last six residues of P11 inserted into a shallow pocket on Stx2A1 and interacted with Arg-172, Arg-176, and Arg-179, which were previously shown to be critical for binding of Stx2A1 to the ribosome. Stx2a formed a distinct P11-binding mode within a different surface pocket relative to ricin toxin A subunit and trichosanthin, suggesting different ribosome recognition mechanisms for each ribosome inactivating protein (RIP). The binding mode of Stx2a to P11 is also conserved among the different Stx subtypes. Furthermore, the P stalk protein CTD is flexible and adopts distinct orientations and interaction modes depending on the structural differences between the RIPs. Structural characterization of the Stx2a-ribosome complex is important for understanding the role of the stalk in toxin recruitment to the sarcin/ricin loop and may provide a new target for inhibitor discovery.

Trichosanthin increases Granzyme B penetration into tumor cells by upregulation of CI-MPR on the cell surface.

Trichosanthin is a plant toxin belonging to the family of ribosome-inactivating proteins. It has various biological and pharmacological activities, including anti-tumor and immunoregulatory effects. In this study, we explored the potential medicinal applications of trichosanthin in cancer immunotherapy. We found that trichosanthin and cation-independent mannose-6-phosphate receptor competitively bind to the Golgi-localized, γ-ear containing and Arf-binding proteins. It in turn promotes the translocation of cation-independent mannose-6-phosphate receptor from the cytosol to the plasma membrane, which is a receptor of Granzyme B. The upregulation of this receptor on the tumor cell surface increased the cell permeability to Granzyme B, and the latter is one of the major factors of cytotoxic T lymphocyte-mediated tumor cell apoptosis. These results suggest a novel potential application of trichosanthin and shed light on its anti-tumor immunotherapy.

A novel trichosanthin fusion protein with increased cytotoxicity to tumor cells.

OBJECTIVE: To evaluate the anti-tumor effects of trichosanthin after fusion with a cell penetrating peptide, heparin-binding peptide (HBP), derived from human heparin-binding EGF-like growth factor (HB-EGF). RESULTS: The fusion protein of trichosanthin-HBP was expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography. The HBP domain had no influence on the topological inactivation activity and N-glycosidase activity of trichosanthin. Trichosanthin-HBP significantly inhibited the growth of tested cancer cells which are impervious to trichosanthin. Tumor cell apoptosis and both the mitochondrial- and death receptor-mediated apoptotic signaling pathways induced by trichosanthin-HBP were more significant than those induced by trichosanthin in HeLa cells. CONCLUSION: HBP is an efficient intracellular delivery vehicle for trichosanthin and makes trichosanthin-HBP become a promising agent for cancer therapy.

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 Šaway from the dimerization domains. (15)N 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.

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.

[Construction, expression and purification of trichosanthin mutant gene TCS(FYY163-165CSA);].

AIM: To construct and express a trichosanthin (TCS) gene mutant and purify the expressed product in E.coli. METHODS: The potential antigenic determinant was predicted on TCS molecule by computer modeling and induced for site-directed mutation. The gene mutant TCS(FYY163-165CSA); was amplified by PCR using the genomic DNA of Trichosanthes kirilowii as a template and cloned into expression vector pRSET-A, then transfected into E.coli BL21 (DE3) for expression by inducing with IPTG. The expressed product was identified by Western blotting and purified by Ni-NTA affinity column chromatography. RESULTS: The soluble target protein was successfully expressed in E.coli. Homogenous TCS mutant protein was obtained after purification of expressed product. CONCLUSION: The site-directed mutagenesis, expression and purification of TCS provide a new approach for reconstructing TCS.

The anti-viral protein of trichosanthin penetrates into human immunodeficiency virus type 1.

Trichosanthin (TCS) is a type I ribosome-inactivating protein with potent inhibitory activity against human immunodeficiency virus type 1, and has been clinically applied in acquired immunodeficiency syndrome (AIDS) therapy. Previous studies revealed that TCS recognized human immunodeficiency virus type 1 (HIV-1) particles. Here, we investigated the physical relationship between TCS and HIV-1 particles, and demonstrated that TCS penetrates into viral particles, where it is protected from various protease digestion. The penetration of TCS exerts no obvious effect on viral integrity. FYY140-142, D176, and K177 were identified as key amino acid residues for the membranetranslocation process. Moreover, TCS penetrated into HIV-1 virions showed potent anti-viral activity. Overall, the observations suggest that the penetration of TCS into HIV-1 particles may be important for eliminating the virus.

Low-density lipoprotein receptor-related protein 1 is an essential receptor for trichosanthin in 2 choriocarcinoma cell lines.

Type-I ribosome-inactivating protein-trichosanthin (TCS) exhibits selective cytotoxicity toward different types of cells. It is believed that the cytotoxicity results from the inhibition of ribosomes to decrease protein synthesis, thereby indicating that there are specific mechanisms for TCS entry into target cells to reach the ribosomes. Low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) is a large scavenger receptor that is responsible for the binding and endocytosis of diverse biological ligands on the cell surface. In this study, we demonstrated that 2 choriocarcinoma cell lines can significantly bind and internalize TCS. In contrast, Hela cell line displayed no obvious TCS binding and endocytosis. Furthermore LRP1 gene silencing in JAR and BeWo cell lines blocked TCS binding; TCS could also interact with LRP1.The results of our study established that LRP1 was a major receptor for phagocytosis of TCS in JAR and BeWo cell lines and might be the molecular basis of TCS abortificient and anti-choriocarcinoma activity.

Retrograde transport of a traditional Chinese medicine, alpha-trichosanthin, and its selective neural toxicity.

OBJECTIVE: To investigate the peripheral neuronal toxicity of a traditional Chinese medicine, alpha-trichosanthin (TCS). METHODS: TCS and rhodamine-conjugated TCS were separately injected into the rat sciatic nerve. Saline and rhodamine were used alone and separately as control solutions. The motor neurons in the spinal cord and sensory neurons in the dorsal root ganglia were separately counted. The entry of TCS molecules into neurons was observed under the fluorescence microscope. The glial reactions were studied by lectin staining and immunohistochemical method. The muscles innervated by the sciatic nerve and distal to the injection sites, and the nerves proximal to the injection sites were also collected and examined. RESULTS: TCS was taken up and transported by peripheral axons, and at a dose of 1 nmol, killed more than 90% of the motor neurons in 5 days, but only one-third of the sensory neurons of the injected nerve. The loss of neurons was permanent, while the increase of glial activities was mild and transient. CONCLUSION: TCS is retrogradely transported by axons of the injected nerve. TCS shows a selective neurotoxicity on different types of neurons. Hence TCS is useful in producing neural lesion in research, and this use may also be of applicational value in treating chronic spasticity, hyperalgesia, and pain.

Increase in cytosolic calcium maintains plasma membrane integrity through the formation of microtubule ring structure in apoptotic cervical cancer cells induced by trichosanthin.

This study investigates the role of dysregulated cytosolic free calcium ([Ca(2+)]c) homeostasis on microtubule (MT) ring structure in apoptotic cervical cancer (HeLa) cells induced by trichosanthin (TCS), a type I ribosome inactivating protein (RIP). The TCS-induced decrease in cell viability was significantly enhanced in combination with the specific calcium chelator, EGTA-AM. Sequestration of [Ca(2+)]c markedly disrupted the special MT ring structure. Furthermore, TCS tended to increase LDH release, whereas no significant differences were observed until 48 h of the treatment. In contrast, combined addition of EGTA-AM or colchicine (an inhibitor of tubulin polymerization) significantly reinforced LDH release. The data suggest that TCS-elevated [Ca(2+)]c maintains plasma membrane integrity via the formation of the MT ring structure in apoptotic HeLa cells.

A novel sorting strategy of trichosanthin for hijacking human immunodeficiency virus type 1.

Trichosanthin (TCS) is a type I ribosome-inactivating protein that plays dual role of plant toxin and anti-viral peptide. The sorting mechanism of such an exogenous protein is in long pursuit. Here, we examined TCS trafficking in cells expressing the HIV-1 scaffold protein Gag, and we found that TCS preferentially targets the Gag budding sites at plasma membrane or late endosomes depending on cell types. Lipid raft membrane but not the Gag protein mediates the association of TCS with viral components. After Gag budding, TCS is then released in association with the virus-like particles to generate TCS-enriched virions. The resulting TCS-enriched HIV-1 exhibits severely impaired infectivity. Overall, the observations indicate the existence of a unique and elaborate sorting strategy for hijacking HIV-1.

A novel strategy for the invasive toxin: hijacking exosome-mediated intercellular trafficking.

Toxins penetrate mammalian cells through various means. In this study, we report a unique strategy used by trichosanthin (TCS), a plant toxin with ribosome-inactivating activity, to penetrate host cells. We found that in both JAR and K562 cells, endocytosed TCS is incorporated into intraluminal vesicles of the multivesicular body (MVB) and is then secreted in association with these vesicles upon fusion of the MVB with the plasma membrane. The secreted TCS-loaded vesicles secreted by K562 cells move throughout the intercellular space and target syngeneic and specific allogeneic cells. Subsequent internalization permits delivery of the toxin into the cytosol, resulting in ribosomal inactivation and cell death. Thus, our findings provide a novel mechanism by which foreign proteins pass between and penetrate into mammalian cells.

[Antitumor effect of recombinant immunotoxin EGF-TCS in nude mice bearing human hepatocellular carcinoma].

OBJECTIVE: To evaluate the anti-tumor effect of recombinant toxin EGF-TCS against transplanted human hepatocellular carcinoma in nude mice. METHODS: Human hepatocellular carcinoma BEL-7,402 cells were inoculated subcutaneously in the right axillary region of nude mice, and 6 days later, EGF-TCS was injected intravenously at 100, 50, and 25 microg/kg. The mice were executed on the next day of drug withdrawal and the tumors were weighed and the tumor inhibition rate calculated. Immunohistochemistry was also performed on the tumor tissues to provide clue for the possible pathways of tumor inhibition. RESULTS: EGF-TCS markedly inhibited the tumor growth in nude mice, with a tumor inhibition rate of 71.3%, 60.87% and 45.22% corresponding to EGF-TCS dosage of 100, 50, and 25 microg/kg, respectively. Variance analysis suggested that EGF-Linker-TCS could significantly inhibit the tumor growth in the mice (F=8.712, P=0.006), and immunohistochemistry showed significantly inhibited angiogenesis in the tumors by EGF-TCS. No blood vessels were found in the tumor tissues in high dosage group, and there were also reduced blood vessels in the other two smaller dose groups in comparison with the untreated model group, indicating that EGF-TCS inhibited tumor growth and migration by inhibiting tumor angiogenesis. CONCLUSION: EGF-TCS can inhibit the growth of solid tumors in nude mice, suggesting the potential value of this preparation in cancer therapy.

[Gene cloning, expression and purification of fusion protein epidermal growth factor-linker-trichosanthin].

OBJECTIVE: To construct a recombinant expression vector of the fusion protein epidermal growth factor (EGF)-Linker-trichosanthin (TCS) and achieve its expression in E. coli to obtain purified EGF-linker-TCS fusion protein. METHODS: The gene fragments of EGF-linker were amplified by PCR and inserted into the expression plasmid PQE30-TCS, followed by transformation of the recombinant plasmid into E. coli M15 for expression of the fusion protein. Ni-FF column chromatography was utilized for purification of the expressed product. RESULTS: The recombinant plasmid PQE30-EGF-linker-TCS was stably and highly expressed in E. coli M15. The expressed product existed in the form of soluble protein accounting for about 40% of total cellular protein and reached a purity of above 95% after purification with Ni-FF column chromatography. CONCLUSION: The recombinant plasmid PQE30/EGF-linker-TCS has been successfully constructed, which provides a basis for further structural and functional study of EGF and TCS and their potential clinical application for cancer therapy.

Two novel proteins bind specifically to trichosanthin on choriocarcinoma cell membrane.

Trichosanthin is the active protein component in the Chinese herb Trichosanthes kirilowi, which has distinct pharmacological properties. The cytotoxicity of trichosanthin was demonstrated by its selective inhibition of various choriocarcinoma cells. When Jar cells were treated with trichosanthin, the influx of calcium into the cells was observed by confocal laser scanning microscopy. When the distribution of trichosanthin-binding proteins on Jar cells was studied, two classes of binding sites for trichosanthin were shown by radioligand binding assay. Furthermore, the cytoplasmic membrane of Jar cells was biotinylated and the trichosanthin-binding proteins were isolated with trichosanthin-coupled Sepharose beads. Two protein bands with molecular masses of about 50 kDa and 60 kDa were revealed, further characterization of which should shed light on the mechanism of the selective cytotoxicity of trichosanthin to Jar cells.

Ribosomal protein L10a, a bridge between trichosanthin and the ribosome.

Trichosanthin is a type I ribosome-inactivating protein with many pharmacological activities. The trichosanthin-coupled Sepharose affinity purification revealed a protein, which was identified by mass spectrometry as the ribosomal protein L10a. The interaction between trichosanthin and recombinant L10a was further confirmed by in vitro binding assay. Kinetic analysis by surface plasmon resonance technology revealed that L10a had a high affinity to trichosanthin with a K(D) of 7.78nM. The study with mutated forms of trichosanthin demonstrated that this specific association correlates with the ribosome-inactivating activity of trichosanthin. This finding might provide insight into the mechanisms by which trichosanthin inactivates ribosome and that underlies its pharmacological effect.

Enhanced green fluorescence protein tracks trichosanthin in human choriocarcinoma cells as a feasible and stable reporter.

Trichosanthin (TCS) is a ribosome-inactivating protein (RIP) which can inhibit the growth of human choriocarcinoma (JAR) cells. There are no clear mechanisms to discover the interaction pathway and cytotoxicity of TCS in JAR cells. In this paper, we showed the distribution and transport of endogenously expressed TCS in JAR cells. Enhanced Green Fluorescence Protein (EGFP), fused with TCS, was applied as a reporter to track the behavior of TCS in JAR cells. Firstly, we investigated the expression stability of EGFP and physiological effects on JAR cells. A stable cell line expressing EGFP was created, which could reproduce and express EGFP even if transplanted into nude mice. Based on the proved stability and feasibility of EGFP in cultured cells and in vivo, the fusion gene of EGFP and TCS was constructed and transfected into JAR cells by liposome. The fluorescence microscopy showed that TCS-EGFP fusion gene was expressed in JAR cells in 24 to 48 hours and the fluorescence spread in cytoplasm mainly and in nucleus partially, which could trace the distribution and transport of TCS-EGFP in JAR cells. Most of fluorescent cells died after 48 hours for the cytotoxicity of expressed TCS-EGFP. These results first reported a stable expression and tracing method by EGFP in JAR cells, and provided theoretical basis to apply TCS in cancer therapy.

Recent advances in trichosanthin, a ribosome-inactivating protein with multiple pharmacological properties.

Trichosanthin (TCS), a ribosome-inactivating protein extracted from the root tuber of Chinese medicinal herb Trichosanthes kirilowii Maximowicz, has multiple pharmacological properties including abortifacient, anti-tumor and anti-HIV. It is traditionally used to induce abortion but its antigenicity and short plasma half-life have limited the repeated clinical administration. In this review, work to locating antigenic sites and prolonging plasma half-life are discussed. Studies on structure-function relationship and mechanism of cell entry are also covered. Recently, TCS has been found to induce apoptosis, enhance the action of chemokines and inhibit HIV-1 integrase. These findings give new insights on the pharmacological properties of TCS and other members of ribosome-inactivating proteins.

Substrate binding and catalysis in trichosanthin occur in different sites as revealed by the complex structures of several E85 mutants.

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) which possesses rRNA N-glycosidase activity. In recent years, its immunomodulatory, anti-tumor and anti-HIV properties have been revealed. Here we report the crystal structures of several E85 mutant TCS complexes with adenosine-5'-monophosphate (AMP) and adenine. In E85Q TCS/AMP and E85A TCS/AMP, near the active site of the molecule and parallel to the aromatic ring of Tyr70, an AMP molecule is bound to the mutant without being hydrolyzed. In the E85R TCS/adenine complex, the hydrolyzed product adenine is located in the active pocket where it occupies a position similar to that in the TCS/NADPH complex. Significantly, AMP is bound in a position different to that of adenine. In comparison with these structures, we suggest that there are at least two subsites in the active site of TCS, one for initial substrate recognition as revealed by the AMP site and another for catalysis as represented by the NADPH site. Based on these complex structures, the function of residue 85 and the mechanism of catalysis are proposed.