A Sixty-Year Research and Development of Trichosanthin, a Ribosome-Inactivating Protein.

Tian Hua Fen, a herbal powder extract that contains trichosanthin (TCS), was used as an abortifacient in traditional Chinese medicine. In 1972, TCS was purified to alleviate the side effects. Because of its clinical applications, TCS became one of the most active research areas in the 1960s to the 1980s in China. These include obtaining the sequence information in the 1980s and the crystal structure in 1995. The replication block of TCS on human immunodeficiency virus in lymphocytes and macrophages was found in 1989 and started a new chapter of its development. Clinical studies were subsequently conducted. TCS was also found to have the potential for gastric and colorectal cancer treatment. Studies on its mechanism showed TCS acts as an rRNA N-glycosylase (EC 3.2.2.22) by hydrolyzing and depurinating A-4324 in α-sarcin/ricin loop on 28S rRNA of rat ribosome. Its interaction with acidic ribosomal stalk proteins was revealed in 2007, and its trafficking in mammalian cells was elucidated in the 2000s. The adverse drug reactions, such as inducing immune responses, short plasma half-life, and non-specificity, somehow became the obstacles to its usage. Immunotoxins, sequence modification, or coupling with polyethylene glycerol and dextran were developed to improve the pharmacological properties. TCS has nicely shown the scientific basis of traditional Chinese medicine and how its research and development have expanded the knowledge and applications of ribosome-inactivating proteins.

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.

Clinical Use of Toxic Proteins and Peptides from Tian Hua Fen and Scorpion Venom.

Traditional Chinese Medicine (TCM) has been practiced in China for thousands of years. As a complementary and alternative treatment, herbal medicines that are frequently used in the TCM are the most accepted in the Western world. However, animal materials, which are equally important in the TCM practice, are not well-known in other countries. On the other hand, the Chinese doctors had documented the toxic profiles of hundreds of animals and plants thousand years ago. Furthermore, they saw the potential benefits of these materials and used their toxic properties to treat a wide variety of diseases, such as heavy pain and cancer. Since the 50s of the last century, efforts of the Chinese government and societies to modernize TCM have achieved tremendous scientific results in both laboratory and clinic. A number of toxic proteins have been isolated and their functions identified. Although most of the literature was written in Chinese, this review provide a summary, in English, regarding our knowledge of the clinical use of the toxic proteins isolated from a plant, Tian Hua Fen, and an animal, scorpion, both of which are famous toxic prescriptions in TCM.

Structural and Functional Investigation and Pharmacological Mechanism of Trichosanthin, a Type 1 Ribosome-Inactivating Protein.

Trichosanthin (TCS) is an RNA N-glycosidase that depurinates adenine-4324 in the conserved α-sarcin/ricin loop (α-SRL) of rat 28 S ribosomal RNA (rRNA). TCS has only one chain, and is classified as type 1 ribosome-inactivating protein (RIP). Our structural studies revealed that TCS consists of two domains, with five conserved catalytic residues Tyr70, Tyr111, Glu160, Arg163 and Phe192 at the active cleft formed between them. We also found that the structural requirements of TCS to interact with the ribosomal stalk protein P2 C-terminal tail. The structural analyses suggest TCS attacks ribosomes by first binding to the C-terminal domain of ribosomal P protein. TCS exhibits a broad spectrum of biological and pharmacological activities including anti-tumor, anti-virus, and immune regulatory activities. This review summarizes an updated knowledge in the structural and functional studies and the mechanism of its multiple pharmacological effects.

Gualou Guizhi decoction reverses brain damage with cerebral ischemic stroke, multi-component directed multi-target to screen calcium-overload inhibitors using combination of molecular docking and protein-protein docking.

Stroke is a disease of the leading causes of mortality and disability across the world, but the benefits of drugs curative effects look less compelling, intracellular calcium overload is considered to be a key pathologic factor for ischemic stroke. Gualou Guizhi decoction (GLGZD), a classical Chinese medicine compound prescription, it has been used to human clinical therapy of sequela of cerebral ischemia stroke for 10 years. This work investigated the GLGZD improved prescription against intracellular calcium overload could decreased the concentration of [Ca2+]i in cortex and striatum neurone of MCAO rats. GLGZD contains Trichosanthin and various small molecular that they are the potential active ingredients directed against NR2A, NR2B, FKBP12 and Calnodulin target proteins/enzyme have been screened by computer simulation. "Multicomponent systems" is capable to create pharmacological superposition effects. The Chinese medicine compound prescriptions could be considered as promising sources of candidates for discovery new agents.

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.

Prodrug-Like, PEGylated Protein Toxin Trichosanthin for Reversal of Chemoresistance.

Multidrug resistance (MDR) is a main obstacle in cancer chemotherapy. The MDR mechanisms involve P-glycoprotein (P-gp) overexpression, abnormality of apoptosis-related protein, and altered expression of drug-targeting proteins. Therapeutic proteins are emerging as candidates for overcoming cancer MDR because of not only their large molecular size that potentially circumvents the P-gp-mediated drug efflux but also their distinctive bioactivity distinguished from small-molecular drugs. Herein we report trichosanthin, a plant protein toxin, possesses synergistic effect with paclitaxel (PTX) in the PTX-resistance A549/T nonsmall cell lung cancer (NSCLC) cells, by reversing PTX-caused caspase 9 phosphorylation and inducing caspase 3-dependent apoptosis. Moreover, via intein-mediated site-specific protein ligation, a matrix metalloproteinase (MMP)-activatable cell-penetrating trichosanthin delivery system was constructed by modification of a cell-penetrating peptide and MMP-2-sensitive PEGylation to overcome the limitation of in vivo application of trichosanthin, by improving the short half-life and poor tumor targeting, as well as immunogenicity. In a mouse model bearing A549/T tumor, the MMP-activatable trichosanthin was further tested for its application for MDR reversal in combination with PTX liposomes. The delivery system showed synergy effect with PTX-loaded liposome in treating MDR cancer in vivo.

Crystal Structure of Ribosome-Inactivating Protein Ricin A Chain in Complex with the C-Terminal Peptide of the Ribosomal Stalk Protein P2.

Ricin is a type 2 ribosome-inactivating protein (RIP), containing a catalytic A chain and a lectin-like B chain. It inhibits protein synthesis by depurinating the N-glycosidic bond at α-sarcin/ricin loop (SRL) of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation center of the ribosome. Here, we present the 1.6 Å crystal structure of Ricin A chain (RTA) complexed to the C-terminal peptide of the ribosomal stalk protein P2, which plays a crucial role in specific recognition of elongation factors and recruitment of eukaryote-specific RIPs to the ribosomes. Our structure reveals that the C-terminal GFGLFD motif of P2 peptide is inserted into a hydrophobic pocket of RTA, while the interaction assays demonstrate the structurally untraced SDDDM motif of P2 peptide contributes to the interaction with RTA. This interaction mode of RTA and P protein is in contrast to that with trichosanthin (TCS), Shiga-toxin (Stx) and the active form of maize RIP (MOD), implying the flexibility of the P2 peptide-RIP interaction, for the latter to gain access to ribosome.

Trichosanthin suppresses the proliferation of glioma cells by inhibiting LGR5 expression and the Wnt/ß-catenin signaling pathway.

Studies have indicated that trichosanthin (TCS), a bioactive protein extracted and purified from the tuberous root of Trichosanthes kirilowii (a well­known traditional Chinese medicinal plant), produces antitumor effects on various types of cancer cells. However, the effects of TCS on glioma cells are poorly understood. The objective of this study was to investigate the antitumor effects of TCS on the U87 and U251 cell lines. The in vitro effects of TCS on these two cell lines were determined using a Cell Counting Kit­8 (CCK­8) assay, Annexin V­FITC staining, DAPI staining, Transwell assays, terminal deoxynucleotidyl transferase­mediated dUTP nick end­labeling (TUNEL) assays, 5,5',6,6'­tetrachloro­1,1',3,3'­tetraethyl­imidacarbocyanine iodide (JC­1) staining and western blotting, which was utilized to assess the expression of leucine­rich repeat­containing G protein­coupled receptor 5 (LGR5) and key proteins in the Wnt/ß­catenin signaling pathway. Our data indicated that TCS inhibited the proliferation of glioma cells in a dose­ and time­dependent manner and played a role in inhibiting glioma cell invasion and migration. Additional investigation revealed that the expression levels of LGR5 and of key proteins in the Wnt/ß­catenin signaling pathway were markedly decreased after TCS treatment. The results suggest that TCS may induce apoptosis in glioma cells by targeting LGR5 and repressing the Wnt/ß­catenin signaling pathway. In the future, in vivo experiments should be conducted to examine the potential use of this compound as a novel therapeutic agent for gliomas.

Structures of eukaryotic ribosomal stalk proteins and its complex with trichosanthin, and their implications in recruiting ribosome-inactivating proteins to the ribosomes.

Ribosome-inactivating proteins (RIP) are RNA N-glycosidases that inactivate ribosomes by specifically depurinating a conserved adenine residue at the α-sarcin/ricin loop of 28S rRNA. Recent studies have pointed to the involvement of the C-terminal domain of the eukaryotic stalk proteins in facilitating the toxic action of RIPs. This review highlights how structural studies of eukaryotic stalk proteins provide insights into the recruitment of RIPs to the ribosomes. Since the C-terminal domain of eukaryotic stalk proteins is involved in specific recognition of elongation factors and some eukaryote-specific RIPs (e.g., trichosanthin and ricin), we postulate that these RIPs may have evolved to hijack the translation-factor-recruiting function of ribosomal stalk in reaching their target site of rRNA.

A Trichosanthin-derived peptide suppresses type 1 immune responses by TLR2-dependent activation of CD8(+)CD28(-) Tregs.

A group of 15-aa-long Trichosanthin-derived peptides was synthesized and screened based on their differential abilities to induce low-responsiveness in mouse strains with high and low susceptibility. One of them was conjugated to form a homo-tetramer Tk-tPN. At concentrations of 0.1-50 µg/ml, Tk-tPN activated CD8(+)CD28(-) Tregs in vitro to induce immune suppression as effectively as the native Trichosanthin but did not exhibit cytotoxicity. In EAE mice which were pre-treated with Tk-tPN or Tk-tPN-activated CD8(+) T cells, a marked attenuation of clinical scores was recorded together with an expansion of the CD8(+)CD28(-) Treg from 2.2% to 36.1% in vivo. A pull-down assay and signal transduction analyses indicated that the ability of Tk-tPN to convert the CD8(+)CD28(-) Treg-related cytokine secretion pattern from type 1 to type 2 depends on the TLR2-initiated signaling in macrophages. The high production of IL-4/IL-10 by the Tk-tPN-activated CD8(+)CD28(-) Treg suggests the value of using Tk-tPN as a therapeutic reagent for Th1-dominant immunological diseases.

Possible mechanisms of trichosanthin-induced apoptosis of tumor cells.

Trichosanthin (TCS) is a type I ribosome-inactivating protein that is isolated from the root tubers of the Chinese medicinal herb Trichosanthes kirilowii Maximowicz. TCS has been used as an abortifacient for 1,500 years in China because of its high toxicity on trophoblasts. Over the past 20 years, TCS has been the subject of much research because of its potential antitumor activities. Many reports have revealed that TCS is cytotoxic in a variety of tumor cell lines in vitro and in vivo. Monoclonal antibody-conjugated TCS could enhance its antitumor efficacy; thus, TCS is considered to be a potential biological agent for cancer treatment. TCS is able to inhibit protein synthesis and consequently induce necrosis. Recent studies have demonstrated that TCS does indeed induce apoptosis in several tumor cell lines. Although TCS-induced apoptosis of tumor cell lines is now well known, the underlying mechanisms remain to be elucidated. The purpose of this review was to investigate the effects of TCS and its possible mechanisms of action, based on published literature and the results of our own studies.

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 A 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.

[A comparative study on the properties of trichosanthin before and after site-directed PEGylation].

OBJECTIVE: To construct PEGylated trichosanthin (TCS) mutein and analyze its bioactivities, immunogenicity, acute toxicity, and pharmacokinetics. METHODS: The potential antigenic determinant site YFF81-83 in the molecule of TCS was selected to undergo site-directed mutagenesis. Thus, a TCS mutein named TCS(YFF81-83ACS) was constructed and expressed in Escherichia coli of the line BL21 (DE3). Wild TCS (wTCS), TCSY(FF81-83ACS), and PEGylated TCS(YFF81-83ACS) (PEG- TCS(YFF81-83ACS)) of different concentrations were incubated with the supercoiled plasmid pUC19 to detect the DNAse activity, mixed with rabbit reticulocyte lysate to detect the ribosome inactivation activity, subcutaneously injected into 6 mice respectively to measure the serum IgG and IgE levels, intravenously injected into mice to observe the toxicity, and intravenously injected into SD rats to observe its -plasma half-life. RESULTS: The DNAse activity of the PEG-TCS(YFF81-83ACS) was similar to that of the wTCS. The ribosome inactivation activity of the PEG-TCS(YFF81-83ACS) was 1/9-1/8 of that of the wTCS (P < 0.05). The serum IgE and IgG levels of the PEG-TCS(YFF81-83ACS) were both significantly lower than those of the wTCS (both P < 0.05). The LD50 of the PEG-TCS(YFF81-83ACS) was 1.8 times that of the wTCS (P < 0.05). The mean residence time and plasma half-life of the PEG-TCS(YFF81-83ACS) were significantly increased and its plasma clearance was significantly decreased (all P < 0.05). CONCLUSION: Site-directed mutagenesis and PEGylation of TCS provide a new approach for reconstructing TCS.

The C-terminal end of P proteins mediates ribosome inactivation by trichosanthin but does not affect the pokeweed antiviral protein activity.

Ribosome inactivating proteins (RIPs) inhibit protein synthesis depurinating a conserved residue in the sarcin/ricin loop of ribosomes. Some RIPs are only active against eukaryotic ribosomes, but other RIPs inactivate with similar efficiency prokaryotic and eukaryotic ribosomes, suggesting that different RIPs would interact with different proteins. The SRL in Trypanosoma cruzi ribosomes is located on a 178b RNA molecule named 28Sdelta. In addition, T. cruzi ribosomes are remarkably resistant to TCS. In spite of these peculiarities, we show that TCS specifically depurinate the predicted A(51) residue on 28Sdelta. We also demonstrated that the C-terminal end of ribosomal P proteins is needed for full activity of the toxin. In contrast to TCS, PAP inactivated efficiently T.cruzi ribosomes, and most importantly, does not require from the C-terminal end of P proteins. These results could explain, at least partially, the different selectivity of these toxins against prokaryotic and eukaryotic ribosomes.

Different neuronal toxicity of single-chain ribosome-inactivating proteins on the rat retina.

OBJECTIVE: To investigate the neurotoxicity of two structurally similar single chains of ribosome-inactivating proteins (RIPs): trichosanthin (TCS) and ricin A chain (RTA). METHODS: TCS, RTA and Ricinus communis agglutinin (RCA, a multi-chain RIP for comparison) were separately injected into rat eyes. Saline was used as control. The data on cell counts, retinal thickness and histopathological scores were collected, and the TUNEL method (terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling) was used to study the mode of cell death. RESULTS: TCS caused distinct retinal changes at 1 nmol. Its toxic effects were most pronounced on the cells of the outer nuclear layer, less so on those of the inner nuclear layer, and little on the ganglion cells. Apoptosis was the predominant type of cell death induced by TCS. In contrast, RTA and RCA, both at 0.01 nmol, brought about acute retinal inflammation and necrosis. CONCLUSION: TCS can eliminate specific retinal cells by apoptosis, while RTA and RCA cause retinitis. The B chain of type II RIPs is not obligatory for their neurotoxicity. The RIPs may be useful for creating retinal models and TCS may be useful for the chemical treatment of retinoblastoma.

Trichosanthin down-regulated p210Bcr-Abl and enhanced imatinib-induced growth arrest in chronic myelogenous leukemia cell line K562.

PURPOSE: Trichosanthin (TCS), an active component extracted from the root tubers of traditional Chinese medical herb Tian-Hua-Fen of the Cucurbitaceae family, has long been used for medical purpose in China; there is increasing interest in developing TCS as cancer therapeutic agents. The present study was to investigate the growth arrest of K562 cells and its molecular mechanisms, which the drugs induced by TCS and the possible functional interaction of TCS with imatinib (STI571) to K562 cells. METHODS: Trypan blue exclusive staining was used to access the cell growth inhibition; western blot was used to evaluate the p210(Bcr-Abl), phosphorylated tyrosine kinase (PTK), and some signaling molecules involving in cell proliferation and apoptosis in K562 cells. RESULTS: TCS and imatinib inhibited K562 cells at a time- and dose-dependent manners, respectively; TCS down-regulated p210(Bcr-Abl) at a time- and dose-dependent manners; TCS synergistically enhanced imatinib-induced K562 cell growth arrest and down-regulation of p210(Bcr-Abl), PTK activities, procaspase-3, Hsp90,NF-kappaB and PKC. CONCLUSION: The results suggest that TCS not only by itself involves but also synergizes activities of imatinib to induce K562 cell growth arrest, down-regulation of p210(Bcr-Abl) and its downstream signals and to stimulate the effect of the tyrosine kinase inhibition.

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 A(4324) at the alpha-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.

Role of the C-terminal region of the B component of Methylosinus trichosporium OB3b methane monooxygenase in the regulation of oxygen activation.

The effects of the C-terminal region of the B component (MMOB) of soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b on steady-state turnover, the transient kinetics of the reaction cycle, and the properties of the sMMO hydroxylase (MMOH) active site diiron cluster have been explored. MMOB is known to have many profound effects on the rate and specificity of sMMO. Past studies have revealed specific roles for the well-folded core structure of MMOB as well as the disordered N-terminal region. Here, it is shown that the disordered C-terminal region of MMOB also performs critical roles in the regulation of catalysis. Deletion mutants of MMOB missing 5, 8, and 13 C-terminal residues cause progressive decreases in the maximum steady-state turnover number, as well as lower apparent rate constants for formation of the key reaction cycle intermediate, compound Q. It is shown that this latter effect is actually due to a decrease in the rate constant for formation of an earlier intermediate, probably the hydroperoxo species, compound P. Moreover, the deletions result in substantial uncoupling at or before the P intermediate. It is proposed that this is due to competition between slow H(2)O(2) release from one of the intermediates and the reaction that carries this intermediate on to the next step in the cycle, which is slowed by the mutation. Electron paramagnetic resonance (EPR) studies of the hydroxylase component (MMOH) in the mixed valent state suggest that complexation with the mutant MMOBs alters the electronic properties of the diiron cluster in a manner distinct from that observed when wild-type MMOB is used. Active site structural changes are also suggested by a substantial decrease in the deuterium kinetic isotope effect for the reaction of Q with methane thought to be associated with a decrease in quantum tunneling in the C-H bond breaking reaction. Thus, the surface interactions between MMOH and MMOB that affect substrate oxidation and its regulation appear to require the complete MMOB C-terminal region for proper function.