The structural basis of Trp192 and the C-terminal region in trichosanthin for activity and conformational stability

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) possessing N-glycosidase activity. TCS has various pharmacological properties, including immunomodulatory, anti-tumor and anti-HIV activities. Up to seven C-terminal residues of TCS (TCS-C7) can be deleted resulting in lower antigenicity with minimal effects on its activity. However, an additional problem is that the minimal effects on activity are higher than the reduction in antigenicity. In the present work, the crystal structure of TCS-C7 was determined. It shows the details of the C-terminal residues of TCS-C7, and in particular the hydrogen bonds between P35 and L240, S196 and L240, and W192 and L239, which play an important role in maintaining the structure of TCS-C7. Further analysis shows that the hydrogen bonds related to Leu240 are key in maintaining the relationship between N- and C-terminal domains. The major role of the C-terminal tail appears to stabilize the structure of TCS. The conformation between helix H7 at the N-terminal domain and the C-terminal tail at the C-terminal domain is also revealed. Two mutants, TCS-W192F and TCS-C7-W192F, were prepared and crystal structures were determined. These variants have greatly reduced ribosome-inactivating activities compared with TCS and TCS-C7, respectively, and TCS-W192F and TCS-C7-W192F have a similar stability in guanidine hydrochloride compared with TCS-C7. This suggests that Trp192 can affect the ribosome-inactivating activity of TCS.     

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.     

天花粉蛋白与CibacronBlueF_3GA结合特性的研究

 天花粉蛋白与ＣｉｂａｃｒｏｎＢｌｕｅＦ_３ＧＡ结合特性的研究何贤辉,柯一保,孙汛,聂慧玲（中国科学院上海细胞生物学研究所,上海２０００３１）天花粉蛋白（Ｔｒｉｃｈｏｓａｎｔｈｉｎ,简称ＴＣ８）是从葫芦科植物栝楼（Ｔｂｅｈｏｓａｎｔｈｅｓｋｉｒｉｌｏｗ?..     

Change in pH-dependent membrane insertion characteristics of trichosanthin caused by deletion of its last seven C-terminal amino acid residues

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) that can selectively kill some types of cells at low concentration (0.1-1 nM). The pH-dependent membrane insertion ability of TCS makes it possible that the internalized toxin avoids degradation in lysosomes and further undergoes transportation into the cytosol by some still unidentified mechanism. Here, we show that deletion of C-terminal residues affects interactions of modified TCS (C7-TCS) with lipids and reduces its pH-dependent membrane insertion ability. Fluorescence measurements indicate that at low pH C7-TCS undergoes profound conformational changes that causes exposure of a hydrophobic region and leads to oligomerization of the C7-TCS molecules. The results suggest that the membrane insertion of TCS at low pH might be important for translocation of TCS into the cytosol, which is important for exertion of the RIP activity of TCS. Deletion of the last seven C-terminal residues of TCS would reduce both its RIP activity in vitro and cytotoxicity in vivo, with the degree of decrease being more significant for the cytotoxicity in vivo.     

Effect of site-directed PEGylation of trichosanthin on its biological activity, immunogenicity, and pharmacokinetics

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) with multiple biological and pharmacological activities. It has been approved effective in the clinical treatment of AIDS and tumor, but its strong immunogenicity and short plasma half-life have limited the clinical administration. To reduce the immunogenicity and prolong the plasma half-life of this compound, three TCS muteins (M(1), M(2) and M(3)) and two PEGylated TCS muteins (PM(1) and PM(2)) were constructed by site-directed mutagenesis and PEGylation, respectively. Compared with the unmodified TCS, both PEGylated TCS showed a 3- to 4-fold decrease in immunogenicity, a 0.5- to 0.8-fold decrease in non-specific toxicity, and a 4.5- to 6-fold increase in plasma half-life. But there is a problem of activity reduction. The increased circulating half-life in vivo may compensate for the reduced activity. Together with the other benefits of PEGylation such as reduced immunogenicity and toxicity, it is worthwhile to further explore the potential application of the PEGylated TCS as a better therapeutic agent for AIDS and tumor.     

天花粉蛋白的定点聚乙二醇修饰

用一种定点修饰天花粉蛋白（ｔｒｉｃｈｏｓａｎｔｈｉｎ,ＴＣＳ）的方法,将聚乙二醇（ＰＥＧ）偶联到预先选定的位点．利用ｎＴＣＳ无半胱氨酸（Ｃｙｓ）残基这一特点,通过定点突变将一个Ｃｙｓ残基引入ＴＣＳ以取代第７位的丝氨酸（Ｓｅｒ）残基．然后,与巯基反应的ＰＥＧ－ｍ ａｌｅｉｍ ｉｄｅ 即可偶联到新引入的Ｃｙｓ 残基上．经纯化得到均一的ＰＥＧ－ＴＣＳ复合物,在ＳＤＳ－ＰＡＧＥ上显示一条区带,表观分子量为３８ ｋＤ．复合物的体外致核糖体失活活性降低了６倍,但其体内引产活性与ｎＴＣＳ相同．定点ＰＥＧ修饰方法为改造ＴＣＳ提供了新途径．     

Molecular modeling of the interactions of trichosanthin with four substrate analogs

Trichosanthin (TCS) is a ribosome-inactivating protein (RIP) that possesses N-glycosidase activity. It inactivates ribosomes and arrests protein synthesis by removing a specific adenine from 28S rRNA. A molecular dynamics simulated annealing method was applied to study the binding modes of TCS with substrate analogs, three oligonucleotides GAG, GAGA, and CGAGAG, based on the crystal structures of the stable complexes of TCS with NADPH and with the reaction product adenine. A water molecule proposed to be responsible for hydrolyzing the N-glycosidic bond was included in the model. All the oligoribonucleotides can dock into the active cleft of TCS without unfavorable contacts. The interaction energies between TCS and the three oligonucleotides were calculated. The interactions of TCS with NADH were also studied by a molecular dynamics simulated annealing method. The interaction energy between NADH and TCS was compared with that between NADPH and TCS, showing that the lack of 2-phosphate group leads to an energy rise of 20 kcal/mol.     

Triclosan-lysozyme complex as novel antimicrobial macromolecule: a new potential of lysozyme as phenolic drug-targeting molecule

A novel anti-infection strategy to alleviate antibiotic-resistance problem and non-specific toxicity associated with chemotherapy is explored in this study. It is based on utilizing a bacteriolytic enzyme (lysozyme) as a carrier to allow specific targeting of a potential phenolic antimicrobial drug (triclosan) to microbial cells. Lysozyme (LZ) was complexed, via electrostatic and hydrophobic condensation at alkaline pH, to various degrees with triclosan (TCS), a negatively charged phenolic antimicrobial that inhibits bacterial fatty acid synthesis. Fluorescence and absorbance spectra analysis revealed non-covalent association of TCS with the aromatic residues at the interior of LZ molecule. The conjugation greatly promoted the lytic activity of LZ as the degree of TCS derivatization increased. The complexation with LZ turned TCS into completely soluble in aqueous solution. TCS-LZ complexes showed significantly enhanced bactericidal activity against several strains of Gram-positive and Gram-negative bacteria compared to the activity of TCS or LZ alone when tested at the same molar basis. Strikingly, TCS-LZ complex, but not LZ or TCS alone, exhibited unique specificity to scavenge superoxide radicals, generated by the natural xanthine/xanthine oxidase coupling system, without affecting the catalytic function of oxidase. This finding is the first to describe that the membrane disrupting function of lysozyme can be utilized to specifically target antimicrobial drug(s) to pathogen cells and heralding a fascinating opportunity for the potential candidacy of TCS-LZ as novel antimicrobial strategy for human therapy.     

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.     

Trichosanthin induces leakage and membrane fusion of liposome

Trichosanthin (TCS) is a ribosome inactivating protein with multiple pharmacological properties. Here the interaction between TCS and a phospholipid bilayer is investigated to provide evidence for membrane translocation mechanism of TCS. The results show that TCS can destabilize liposomes made by phospholipids with negatively charged head group. The destabilization effect is pH-dependent and happens only under acidic conditions. Membrane fusion is also seen to accompany the destabilizing process. The interaction between a phospholipid bilayer and C7, a mutant of TCS with 7 residues at its C-terminus deleted, has been investigated. Deleting the C-terminus almost completely abolishes the destabilizing effect of TCS on the phospholipid bilayer, which implicates the C-terminus in the interaction between trichosanthin and the membrane.     

Enhancement of the anti-herpetic effect of trichosanthin by acyclovir and interferon

Trichosanthin (TCS) is a type I ribosome-inactivating protein that has a wide range of pharmacological activities. The present study investigated the effectiveness of TCS on herpes simplex virus (HSV-1). The anti-viral activity and toxicity of TCS on Vero cells were measured. Results showed that the ED(50), TD(50) and the therapeutic indices were 38.5, 416.5 and 10.9 microg/ml, respectively. Anti-viral activity of TCS was substantially potentiated when it was used in conjunction with other anti-viral agents. The ED(50) of TCS was reduced 125-fold by acyclovir at a concentration of 0.001 microg/ml, which was practically devoid of significant anti-viral activity. Similarly, the ED(50) of TCS was reduced 100-fold by interferon-alpha2a at a concentration of 100 IU/ml. In conclusion, TCS is effective against HSV-1 and other anti-viral agents such as acyclovir or interferon can potentiate its action substantially.     

Protonophoric action of triclosan causes calcium efflux from mitochondria,plasma membrane depolarization and bursts of miniature end-plate potentials

The formerly widely used broad-spectrum biocide triclosan (TCS) has now become a subject of special concern due to its accumulation in the environment and emerging diverse toxicity. Despite the common opinion that TCS is an uncoupler of oxidative phosphorylation in mitochondria, there have been so far no studies of protonophoric activity of this biocide on artificial bilayer lipid membranes (BLM). Yet only few works have indicated the relationship between TCS impacts on mitochondria and nerve cell functioning. Here, we for the first time report data on a high protonophoric activity of TCS on planar BLM. TCS proved to be a more effective protonophore on planar BLM, than classical uncouplers. Correlation between a strong depolarizing effect of TCS on bacterial membranes and its bactericidal action on Bacillus subtilis might imply substantial contribution of TCS protonophoric activity to its antimicrobial efficacy. Protonophoric activity of TCS, monitored by proton-dependent mitochondrial swelling, resulted in Ca²⁺ efflux from mitochondria. A comparison of TCS effects on molluscan neurons with those of conventional mitochondrial uncouplers allowed us to ascribe the TCS-induced neuronal depolarization and suppression of excitability to the consequences of mitochondrial deenergization. Also similar to the action of common uncouplers, TCS caused a pronounced increase in frequency of miniature end-plate potentials at neuromuscular junctions. Thus, the TCS-induced mitochondrial uncoupling could alter neuronal function through distortion of Ca²⁺ homeostasis.     

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.     

In vitro effects of triclosan and methyl-triclosan on the marine gastropod Haliotis tuberculata

Triclosan (2,4,4'-trichloro-2'-hydroxy-diphenyl ether; TCS) is an antibacterial agent incorporated in a wide variety of household and personal care products. Because of its partial elimination in sewage treatment plants, TCS is commonly detected in natural waters and sediments. Moreover, due to its high hydrophobicity, TCS accumulates in fatty tissues in various aquatic organisms. TCS can be converted into methyl-triclosan (2,4,4'-trichloro-2'-methoxydiphenyl ether; MTCS) after biological methylation. In this study, the acute cytotoxicity of TCS and MTCS in short-term in vitro experiments was assessed on cell cultures from the European abalone Haliotis tuberculata. The results showed that morphology and density of hemocyte are affected from a concentration of 8 μM TCS. Using the XTT reduction assay, TCS has been demonstrated to decrease hemocyte metabolism activity in a dose- and time-dependent exposure. The IC(50) was evaluated at 6 μM for both hemocyte and gill cells after a 24 h-incubation with TCS. A significant cytotoxicity of MTCS was also observed from 4 μM in 24 h-old hemocyte culture. Our results reveal a toxic effect of TCS and MTCS on immune (hemocytes) and/or respiratory cells (gill cells) of the abalone, species living in coastal waters areas and exposed to anthropogenic pollution.     

Site-directed polyethylene glycol modification of trichosanthin: effects on its biological activities, pharmacokinetics, and antigenicity

Trichosanthin (TCS), a type I ribosome-inactivating protein (RIP), was modified with polyethylene glycol (PEG) in order to reduce its antigenicity and prolong its half-life. Computer modeling identified three potential antigenic sites namely Q219, K173 and S7. By site-directed mutagenesis, these sites were changed into cysteine through which PEG can be covalently attached. The resulting TCS had a PEG coupled directly above one of its potential antigenic determinants, hence masking the antigenic region and prevent binding of antibodies specific to this site. In general, mutation did not bring about significant changes in ribosome-inactivating activity, cytotoxicity, and abortifacient activity of TCS. However, the in vitro activities of PEG modified (PEGylated) TCS muteins were 3-20 folds lower and the in vivo activity 50% less than that of nTCS. Pharmacokinetics study indicated that all three PEGylated TCS muteins showed 6-fold increase in mean residence time as compared to unmodified muteins. The binding affinity of an IgE monoclonal antibody (TE1) to TCS was greatly reduced after PEG modification (PEGylation) at position Q219, suggesting that TE1 recognized an epitope very near to residue Q219. PEGylated TCS muteins induced similar IgG response but 4-16 fold lower IgE response in mice compared with nTCS.     

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.     

Genome-wide identification and expression analysis of two component system genes in Cicer arietinum

The two-component system (TCS) plays an important role in signal transduction pathways, cytokinin signaling and stress resistance of prokaryotes and eukaryotes. It is comprised of three types of proteins in plants; histidine kinases (HKs), histidine phosphotransfer proteins (HPs) and response regulators (RRs). Chickpea (Cicer arietinum L.) is one of the most important legume crops worldwide with special economic value in semi-arid tropics. Availability of complete genome sequence of chickpea presents a valuable resource for comparative analysis among angiosperms. In current study, Arabidopsis thaliana and Oryza sativa were used as reference plant species for comparative genomics analysis with C. arietinum. A genome-wide computational survey enabled us to identify putative members of TCS protein family including 18HKs, 26 RRs (7 type-A, 7 type-B, 2 type C and 10 pseudo) and 7 HPs (5 true and 2pseudo) genes in chickpea. The predicted TCS genes displayed family specific intron/exon organization and were randomly distributed across all the eight chromosomes. Comparative phylogenetic and evolutionary analysis suggested a variable conservation of TCS genes in relation to mono/dicot model plants and segmental duplication was the principal route of expansion for this family in chickpea. The promoter regions of TCS genes exhibited several abiotic stress-related cis-elements indicating their involvement in abiotic stress response. The expression analysis of TCS genes demonstrated stress (drought, heat, osmotic and salt) specific differential expression. Current study provides insight into TCS genes in C. arietinum, which will be helpful for further functional analysis of these genes in response to different abiotic stresses.     

Effects of applying electrical stimulation to the locus coeruleus on neuronal activity in the parietal association cortex

It was shown during experiments on cats undergoing surgery under ketamine-induced anesthesia and immobilized with myorelaxin that applying trains of stimuli to the locus coeruleus (LC) produces an effect on 79% of parietal cortex neurons. This manifests as inhibition lasting 300–700 msec or a 16–32% decline in the activity rate of neurons with background activity. Hyperpolarization of 5–7 mV lasting 120–500 msec preceded by a latency of 30–90 msec was noted in such neurons as well as "silent" cells during intracellular recording. Duration of the inhibitory pause in neuronal background activity induced by transcallosal stimulation (TCS) increased by 50–200 msec under the effects of conditioned stimuli applied to the LC. Duration of the IPSP triggered by TCS likewise increased (by 50–100 msec) under the effects of LC stimulation. It was concluded that the effects of stimulating the LC on neuronal activity in the parietal cortex may manifest either directly, as inhibition of background activity and hyperpolarization, or else as modulation of influences exerted by other neurotransmitters.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 4, pp. 486–494, July–August, 1990.