RESUMO
Osteosarcoma is a kind of primary bone malignant tumors. Its cure rate has been stagnant in the past decade years. Curcin C belongs to type I ribosome inactivating proteins, extracted from the cotyledons of post-germinated Jatropha curcas seeds. It can inhibit the proliferation of several tumor lines including U2OS cells with extraordinary efficiency. The treated U2OS cells were arrested in both S and G2/M phase, showed typical apoptosis morphological characteristic, formed autophagosomes and increase the ratio of LC3II to LC3I. Meanwhile, the level of ROS in the treated cells was found increasing significantly, with the change of mitochondrial membrane potential and decreased antioxidant enzyme activities. The application of ROS scavenger NAC not only significantly inhibited the toxicity of Curcin C but also prevented the happen of apoptosis and autophagy to some extent. These results suggested that Curcin C may function through ROS pathway. In addition, the Curcin C treatment could activate JNK and inhibit ERK signal pathway. Sp600125, an inhibitor of JNK signaling pathway, can prevent subsequent apoptosis and autophagy events, suggesting that JNK pathway was at least one of the pathways of Curcin C action. Moreover, the relevant including antagonistic among autophagy, apoptosis and cell cycle arresting induced by Curcin C also was found. In summary, it can be speculated that Curcin C may induce S, G2/M phase arrest, apoptosis and autophagy of human osteosarcoma U2OS cells through activating JNK signal pathway and blocking ERK signal pathway by promoting ROS accumulation in cell, thus finally reflected in the effect of inhibiting tumor cell proliferation.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Proteínas Inativadoras de Ribossomos Tipo 1/química , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificaçãoRESUMO
Using the pathosystem Phaseolus vulgaris-tobacco necrosis virus (TNV), we demonstrated that PD-L1 and PD-L4, type-1 ribosome inactivating proteins (RIPs) from leaves of Phytolacca dioica L., possess a strong antiviral activity. This activity was exerted both when the RIPs and the virus were inoculated together in the same leaf and when they were inoculated or applied separately in the adaxial and abaxial leaf surfaces. This suggests that virus inhibition would mainly occur inside plant cells at the onset of infection. Histochemical studies showed that both PD-L1 and PD-L4 were not able to induce oxidative burst and cell death in treated leaves, which were instead elicited by inoculation of the virus alone. Furthermore, when RIPs and TNV were inoculated together, no sign of H2O2 deposits and cell death were detectable, indicating that the virus could have been inactivated in a very early stage of infection, before the elicitation of a hypersensitivity reaction. In conclusion, the strong antiviral activity is likely exerted inside host cells as soon the virus disassembles to start translation of the viral genome. This activity is likely directed towards both viral and ribosomal RNA, explaining the almost complete abolition of infection when virus and RIP enter together into the cells.
Assuntos
Antígeno B7-H1/farmacologia , Phaseolus/virologia , Phytolacca/química , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Tombusviridae/efeitos dos fármacos , Antivirais/farmacologia , Antígeno B7-H1/isolamento & purificação , Interações entre Hospedeiro e Microrganismos , Folhas de Planta/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificaçãoRESUMO
Gelonin from the Indian plant Gelonium multiflorum belongs to the type I ribosome-inactivating proteins (RIPs). Like other members of RIPs, this toxin glycoprotein inhibits protein synthesis of eukaryotic cells; hence, it is largely used in the construction of immunotoxins composed of cell-targeted antibodies. Lysosomal degradation is one of the main issues in targeted tumor therapies, especially for type I RIP-based toxins, as they lack the translocation domains. The result is an attenuated cytosolic delivery and a decrease of the antitumor efficacy of these plant-derived toxins; therefore, strategies to permit their release from endosomal vesicles or modifications of the toxins to make them resistant to degradation are necessary to improve their efficacy. Using infrared spectroscopy, we thoroughly analyzed both the secondary structure and the thermal unfolding of gelonin. Moreover, by the combination of two-dimensional correlation spectroscopy and phase diagram method, it was possible to deduce the sequence of events during the unfolding, confirming the typical characteristic of the RIP members to denature in two steps, as a sequential loss of tertiary and secondary structure was detected at 58 °C and at 65 °C, respectively. Additionally, some discrepancies in the unfolding process between gelonin and saporin-S6, another type I RIP protein, were detected.
Assuntos
Temperatura Alta , Desdobramento de Proteína , Proteínas Inativadoras de Ribossomos Tipo 1/química , Suregada/química , Toxinas Biológicas/química , Dicroísmo Circular , Estrutura Secundária de Proteína , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Sementes/química , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Toxinas Biológicas/isolamento & purificaçãoRESUMO
A novel type I ribosome-inactivating protein (RIP), designated as curcin C, was purified from Jatropha curcas, an important feedback source of bio-fuel. Molecular mass and isoelectric point of curcin C were 31.398 kDa and 7.12 as detected by MALTI-TOF assay and capillary electrophoresis assay, respectively. N-terminal sequence and LC-MS/MS analyses confirmed that curcin C is a type I RIP having high homology, but not the exactly the same with curcin, another type 1 RIP isolated from the endosperm of J. curcas. It exhibited N-glycosidase activity and in vitro translation inhibition activity. Moreover, curcin C displayed a strong selectively anti-tumor activity on human cancer cells. Its cytotoxicity against osteosarcoma cell line U20S is even higher than that of Paclitaxel with IC50 of 0.019 µM. Purification and identification of curcin C not only suggested its potential in natural anticancer drug development, but also provide chance to understanding different cytotoxic action among different RIPs.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Cotilédone/química , Jatropha/química , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Sequência de Aminoácidos , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cotilédone/crescimento & desenvolvimento , Cotilédone/metabolismo , Humanos , Concentração Inibidora 50 , Ponto Isoelétrico , Jatropha/crescimento & desenvolvimento , Jatropha/metabolismo , Peso Molecular , Osteoblastos/efeitos dos fármacos , Osteoblastos/patologia , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/química , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificaçãoRESUMO
Triterpenoidal saponins are synthesized in the roots of Saponaria officinalis L. The same plant is also a source for the toxin Saporin, which is a ribosome-inactivating protein. Triterpenoidal saponins are known to increase the cytotoxicity of Saporin by modulating its intracellular trafficking. Here, we investigated if the combinatorial effects elicited by purified saponins and Saporin can be applied to increase the therapeutic efficacy of the immunotoxin Saporin-Rituximab. First, saponins were purified by high-performance liquid chromatography. Thereafter, their intrinsic cytotoxicity was evaluated on Ramos cells with no observed effect up to 5 µg/mL, however, saponins increased the cytotoxicity of Saporin, while no influence was observed on its N-glycosidase activity. Saporin-Rituximab bound to CD20 in Ramos cells and, in the absence of saponins, had a GI50 (concentration inhibiting cell growth to 50â%) of 7 nM. However, in the presence of a nontoxic concentration of saponins, the GI50 of Saporin-Rituximab was 0.01 nM, a nearly 700-fold increase in efficacy. Moreover, two further immunotoxins, namely Saporin-anti-CD22 and Saporin-anti-CD25, were tested in combination with saponins yielding enhancement factors of 170-fold and 25-fold, respectively. All three receptors are present in Ramos cells and the differences in cytotoxicity enhancement may be explained by the differing expression levels of the cellular receptors. The application of purified saponins from S. officinalis L. is therefore a new strategy to potentially improve the cytotoxicity and therapeutic efficacy of Rituximab-immunotoxins for the treatment of B-cell lymphoma.
Assuntos
Imunotoxinas/farmacologia , Linfoma de Células B/patologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Rituximab/farmacologia , Saponaria/química , Saponinas/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Sinergismo Farmacológico , Humanos , Imunotoxinas/química , Imunotoxinas/isolamento & purificação , Proteínas Inativadoras de Ribossomos Tipo 1/química , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Saponinas/química , Saponinas/isolamento & purificação , SaporinasRESUMO
Pokeweed antiviral protein (PAP) belongs to the family of type I ribosomeinactivating proteins (RIPs): Ribotoxins, which function by depurinating the sarcinricin loop of ribosomal RNA. In addition to its antibacterial and antifungal properties, PAP has shown promise in antiviral and targeted tumor therapy owing to its ability to depurinate viral RNA and eukaryotic rRNA. Several PAP genes are differentially expressed across pokeweed tissues, with natively isolated seed forms of PAP exhibiting the greatest cytotoxicity. To help elucidate the molecular basis of increased cytotoxicity of PAP isoenzymes from seeds, the present study used protein sequencing, mass spectroscopy and X-ray crystallography to determine the complete covalent structure and 1.7 Å Xray crystal structure of PAPS1aci isolated from seeds of Asian pokeweed (Phytolacca acinosa). PAPS1aci shares ~95% sequence identity with PAPS1 from P. americana and contains the signature catalytic residues of the RIP superfamily, corresponding to Tyr72, Tyr122, Glu175 and Arg178 in PAPS1aci. A rare proline substitution (Pro174) was identified in the active site of PAPS1aci, which has no effect on catalytic Glu175 positioning or overall activesite topology, yet appears to come at the expense of strained mainchain geometry at the preproline residue Val173. Notably, a rare type of Nglycosylation was detected consisting of NacetylDglucosamine monosaccharide residues linked to Asn10, Asn44 and Asn255 of PAPS1aci. Of note, our modeling studies suggested that the ribosome depurination activity of seed PAPs would be adversely affected by the Nglycosylation of Asn44 and Asn255 with larger and more typical oligosaccharide chains, as they would shield the rRNAbinding sites on the protein. These results, coupled with evidence gathered from the literature, suggest that this type of minimal Nglycosylation in seed PAPs and other type I seed RIPs may serve to enhance cytotoxicity by exploiting receptormediated uptake pathways of seed predators while preserving ribosome affinity and rRNA recognition.
Assuntos
Phytolacca americana/química , Processamento de Proteína Pós-Traducional , Inibidores da Síntese de Proteínas/química , RNA Ribossômico/química , Proteínas Inativadoras de Ribossomos Tipo 1/química , Ribossomos/química , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Glicosilação , Isoenzimas/química , Isoenzimas/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Phytolacca americana/enzimologia , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Inibidores da Síntese de Proteínas/isolamento & purificação , RNA Ribossômico/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Ribossomos/metabolismo , Sementes/química , Sementes/enzimologia , Alinhamento de SequênciaRESUMO
Saporin-S6 is a plant toxin belonging to the type 1 ribosome-inactivating protein (RIP) family. Since it was extracted and isolated from Saponaria officinalis for the first time almost thirty years ago, the protein has been widely studied mainly for its potential applications in anti-tumour and anti-viral infection therapy. Like other RIPs, saporin-S6 is particularly effective in the form of immunotoxin conjugated with monoclonal antibodies and its chemico-physical characteristics made the protein a perfect candidate for the synthesis, development and use of saporin-S6-based chimeric toxins. The high stability of the protein against different denaturing agents has been broadly demonstrated, however, its complete thermal unfolding characterization has not already been performed. In this work we analyse in detail structure, thermostability and unfolding features by means of infrared spectroscopy coupled with two-dimensional correlation spectroscopy. Our data showed that saporin-S6 in solution at neutral pH exhibits a secondary structure analogue to that of the crystal and confirmed its good stability at moderately high temperatures, with a temperature of melting of 58°C. Our results also demonstrated that the thermal unfolding process is non-cooperative and occurs in two steps, and revealed the sequence of the events that take place during the denaturation, showing a higher stability of the N-terminal domain of the protein.
Assuntos
Proteínas Inativadoras de Ribossomos Tipo 1/química , Saponaria/química , Sementes/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Modelos Moleculares , Desnaturação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Desdobramento de Proteína , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Saporinas , Soluções , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
CONTEXT: Saponinum album (SA) is a complex mixture of triterpenoid saponins previously shown to augment the cytotoxicity of the type I ribosome-inactivating protein saporin and an EGF-saporin target toxin that could potentially be used to improve the therapeutic window of targeted toxins. OBJECTIVE: To investigate the augmentative property of SA on saporin and saporin-based immunotoxins (IT) directed against five different cell surface target molecules on human leukemia and lymphoma cells. MATERIALS AND METHODS: After determining the optimum dose of SA for each cell line, the extent of SA-mediated augmentation was established for saporin and five saporin-based ITs using XTT and an annexin V apoptosis assay. Immunospecificity was investigated using three different blocking assays. Dose-scheduling was also investigated using the XTT assay. RESULTS: Uncorrected SA-mediated augmentation ranged at best from 31.5 million-fold to, at worse, 174-fold. However, when the calculated fold-increases were adjusted for the non-immunospecific effects of SA on an off-target IT, the true augmentative effects of SA were found to be largely non-immunospecific. Antibody blocking studies demonstrated that the augmentative effect of SA was only partially immunospecific. Separate exposure of target cells to IT and SA at different times demonstrated that immunospecific augmentation of IT by SA could be achieved but only if cells were exposed to IT first and SA second. CONCLUSIONS: SA significantly, although variably, augments the cytotoxicity of saporin and saporin-based immunotoxins. Concomitant exposure to both IT and SA can result in non-immunospecific cytotoxicity that can be overcome by temporally separating exposure to each.
Assuntos
Apoptose/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Saponinas/farmacologia , Triterpenos/farmacologia , Anticorpos Monoclonais Murinos/farmacologia , Antígenos de Diferenciação de Linfócitos T/efeitos dos fármacos , Antígenos de Diferenciação de Linfócitos T/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/imunologia , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Esquema de Medicação , Citometria de Fluxo , Humanos , Terapia de Alvo Molecular , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteínas Inativadoras de Ribossomos Tipo 1/administração & dosagem , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Saponinas/administração & dosagem , Saponinas/isolamento & purificação , Saporinas , Triterpenos/administração & dosagem , Triterpenos/isolamento & purificaçãoRESUMO
Pachyrhizus erosus seeds have a high protein content and are used in China due to their cytotoxic effect. Here we report the biological and pharmacological activity of the protein extracts from P. erosus seeds. A novel ribosome-inactivating protein, pachyerosin, from P. erosus seeds was successively purified to homogeneity using ammonium sulfate precipitation, DEAE-sepharose FF, and Sephacryl S-200. Pachyerosin showed to be a type I ribosome-inactivating protein with a molecular mass of 29 kDa and an isoelectric point of 9.19. It strongly inhibited protein synthesis of rabbit reticulocyte lysate with an IC50 of 0.37 ng/mL and showed N-glycosidase activity on rat liver ribosomes with an EC50 of 85.9 pM. The N-terminal 27 amino acids of pachyerosin revealed a 60.71% sequence identity with abrin A from the seeds of Abrus precatorius. With the aim of targeting the delivery of pachyerosin, immunotoxin was prepared by conjugating pachyerosin with anti-human AFP monoclonal antibodies SM0736. The immunotoxin pachyerosin-SM0736 efficiently inhibited the growth of the human hepatoma cell line HuH-7 with an IC50 of 0.050 ± 0.004 nM, 2360 times lower than that of pachyerosin and 430 times lower than that of the immunotoxin against human gastric cancer cell line SGC7901. These results imply that pachyerosin may be used as a new promising anticancer agent.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Pachyrhizus/química , Extratos Vegetais/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificação , Humanos , Imunotoxinas/isolamento & purificação , Imunotoxinas/farmacologia , Dados de Sequência Molecular , Peso Molecular , Extratos Vegetais/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Plantas Medicinais , Biossíntese de Proteínas/efeitos dos fármacos , Coelhos , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Sementes/química , Alinhamento de SequênciaRESUMO
PD-S2, type 1 ribosome-inactivating protein from Phytolacca dioica L. seeds, is an N-ß-glycosidase likely involved in plant defence. In this work, we purified and characterized an in vivo proteolytic form of PD-S2, named cutPD-S2. Spectroscopic characterization of cutPD-S2 showed that the proteolytic cleavage between Asn195 and Arg196 does not alter the protein fold, but significantly affects its thermal stability. Most importantly, the proteolytic cleavage induces a 370-fold decrease of PD-S2 capacity of inhibiting in vitro protein biosynthesis. Our data catch the turning point from a typical role of PD-S2 as a defence protein to that of supplier of essential amino acids during seedling development.
Assuntos
Aminoácidos Essenciais/metabolismo , Phytolacca/metabolismo , Biossíntese de Proteínas , Proteólise , Proteínas Inativadoras de Ribossomos Tipo 1/química , Sementes/metabolismo , Germinação , Phytolacca/crescimento & desenvolvimento , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Proteínas Inativadoras de Ribossomos Tipo 1/antagonistas & inibidores , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Ribossomos/metabolismo , Sementes/crescimento & desenvolvimentoRESUMO
BACKGROUND: Jatropha curcas seed is a rich source of oil; however, it can not be utilised for nutritional purposes due to presence of toxic and anti-nutritive compounds. The main objective of the present study was to quantify the toxic phytochemicals present in Indian J. curcas (oil, cake, bio-diesel and glycerol). RESULTS: The amount of phorbol esters is greater in solvent extracted oil (2.8 g kg⻹) than in expeller oil (2.1 g kg⻹). Liquid chromatography-mass spectroscopy analysis of the purified compound from an active extract of oil confirmed the presence of phorbol esters. Similarly, the phorbol esters content is greater in solvent extracted cake (1.1 g kg⻹) than in cake after being expelled (0.8 g kg⻹). The phytate and trypsin inhibitory activity of the cake was found to be 98 g kg⻹ and 8347 TIU g⻹ of cake, respectively. Identification of curcin was achieved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the concentration of curcin was 0.95 g L⻹ of crude concentrate obtained from cake. CONCLUSION: Higher amounts of phorbol esters are present in oil than cake but bio-diesel and glycerol are free of phorbol esters. The other anti-nutritional components such as trypsin inhibitors, phytates and curcin are present in cake, so the cake should be detoxified before being used for animal feed.
Assuntos
Biocombustíveis/análise , Glicerol/química , Resíduos Industriais/análise , Jatropha/química , Compostos Fitoquímicos/análise , Óleos de Plantas/química , Sementes/química , Agricultura/economia , Ração Animal/análise , Ração Animal/economia , Biocombustíveis/economia , Ácidos Graxos/análise , Contaminação de Alimentos , Glicerol/economia , Glicerol/isolamento & purificação , Índia , Resíduos Industriais/economia , Ésteres de Forbol/análise , Ésteres de Forbol/economia , Ésteres de Forbol/isolamento & purificação , Ácido Fítico/análise , Ácido Fítico/economia , Ácido Fítico/isolamento & purificação , Compostos Fitoquímicos/economia , Compostos Fitoquímicos/isolamento & purificação , Óleos de Plantas/economia , Óleos de Plantas/isolamento & purificação , Proteínas Inativadoras de Ribossomos Tipo 1/análise , Proteínas Inativadoras de Ribossomos Tipo 1/economia , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Inibidores da Tripsina/análise , Inibidores da Tripsina/economia , Inibidores da Tripsina/isolamento & purificaçãoRESUMO
Ribosome-inactivating proteins (RIPs) are a family of plant toxins that permanently damage ribosomes and possibly other cellular substrates, thus causing cell death. RIPs are mostly divided in two types: Type 1 RIPs that are single-chain enzymatic proteins, and type 2 RIPs that consist of an active A chain (similar to a type 1 RIP) linked to a B chain with lectin properties. RIP-containing conjugates have been used in many experimental strategies against cancer cells, often showing great efficacy in clinical trials. Saporin-S6, a type 1 RIP extracted from Saponaria officinalis L. seeds, has been extensively utilized to construct anti-cancer conjugates because of its high enzymatic activity, stability and resistance to conjugation procedures, resulting in the efficient killing of target cells. This review summarizes saporin-S6-containing conjugates and their application in cancer therapy, considering in-vitro and in-vivo studies both in animal models and in clinical trials. The review is structured on the basis of the targeting of hematological versus solid tumors and on the antigen recognized on the cell surface.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Imunotoxinas/uso terapêutico , Neoplasias/tratamento farmacológico , Proteínas Inativadoras de Ribossomos Tipo 1/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias/sangue , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imunoconjugados/química , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Imunotoxinas/química , Imunotoxinas/farmacologia , Neoplasias/imunologia , Proteínas Inativadoras de Ribossomos Tipo 1/imunologia , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Saponaria/química , Saporinas , Sementes/químicaRESUMO
The seed of the plant Jatropha curcas contains a toxic protein, designated as curcin, which was purified to apparent homogeneity by the combined use of chromatography on Sephdex G-100. The molecular weight of 28.2 kDa and the pI of 8.54 were determined. The protein was found to be a glycoprotein; the total neutral-surge content was 4.91%. It strongly inhibits the protein synthesis of rabbit reticulocyte lysate, with an IC(50) of 0.42 nM. It was determined by Edman that the sequence of the N-terminal thirty-two amino acids was: A-G/Y-S/K-T/A-P/D-T-L-T-I-T-Y-D-A-T/A-A-D-K-K-N-Y-A-Q-F-I-K-D-L-R-E-A-F/A-G. The isolated curcin had a hemagglutinating activity, when its concentration was more than 7.8 mg/L. The secondary structure of curcin was analyzed by Circular Dichroism (CD) spectrum. The result shows the curcin contains alpha-helix (22.3%), beta-sheet (43.5%), and random coil and corner (34.2%). The results of acute toxicity in mice show that mice oral semi-lethal dose LD(50) was 104.737 +/- 29.447 mg/kg; mice parenteral semi-lethal dose LD(50) was 67.20 +/- 10.445 mg/kg.
Assuntos
Extratos Vegetais/química , Extratos Vegetais/toxicidade , Proteínas Inativadoras de Ribossomos Tipo 1/química , Proteínas Inativadoras de Ribossomos Tipo 1/toxicidade , Sementes/química , Animais , Relação Dose-Resposta a Droga , Dose Letal Mediana , Masculino , Camundongos , Extratos Vegetais/isolamento & purificação , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Taxa de Sobrevida , Testes de ToxicidadeRESUMO
A critical problem in studying ribosome-inactivating proteins (RIPs) lies in the very limited possibility to produce them in heterologous systems. In fact, their inherent toxicity for the producing organism nearly always prevents their recombinant expression. In this study, we designed, expressed and characterized an engineered form of the RIP saporin (SapVSAV), bearing a C-terminal extra sequence that is recognized and bound by the second PDZ domain from murine PTP-BL protein (PDZ2). The co-expression of SapVSAV and PDZ2 in Escherichia coli BL21 cells greatly enhances the production of the toxin in a soluble form. The increase of production was surprisingly not due to protection from bacterial intoxication, but may arise from a stabilization effect of PDZ2 on the toxin molecule during biosynthesis. We found that once purified, SapVSAV is stable but is not toxic to free ribosomes, while it is fully active against human cancer cells. This strategy of co-expression of a toxin moiety and a soluble PDZ domain may represent a new system to increase the production of recombinant toxic proteins and could allow the selection of new extra sequences to target PDZ domains inside specific mammalian cellular domains.
Assuntos
Domínios PDZ , Engenharia de Proteínas/métodos , Inibidores da Síntese de Proteínas/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/genética , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Animais , Linhagem Celular , Escherichia coli/genética , Expressão Gênica , Humanos , Inibidores da Síntese de Proteínas/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Saporinas , TransfecçãoRESUMO
Ribosome inactivating proteins (RIPs) catalyze the hydrolytic depurination of one or more adenosine residues from eukaryotic ribosomes. Depurination of the ribosomal sarcin-ricin tetraloop (GAGA) causes inhibition of protein synthesis and cellular death. We characterized the catalytic properties of saporin-L1 from Saponaria officinalis (soapwort) leaves, and it demonstrated robust activity against defined nucleic acid substrates and mammalian ribosomes. Transition state analogue mimics of small oligonucleotide substrates of saporin-L1 are powerful, slow-onset inhibitors when adenosine is replaced with the transition state mimic 9-deazaadenine-9-methylene-N-hydroxypyrrolidine (DADMeA). Linear, cyclic, and stem-loop oligonucleotide inhibitors containing DADMeA and based on the GAGA sarcin-ricin tetraloop gave slow-onset tight-binding inhibition constants (K(i)*) of 2.3-8.7 nM under physiological conditions and bind up to 40000-fold tighter than RNA substrates. Saporin-L1 inhibition of rabbit reticulocyte translation was protected by these inhibitors. Transition state analogues of saporin-L1 have potential in cancer therapy that employs saporin-L1-linked immunotoxins.
Assuntos
Proteínas Inativadoras de Ribossomos/química , Proteínas Inativadoras de Ribossomos/farmacologia , Ribossomos/efeitos dos fármacos , Animais , Morte Celular , Cromatografia Líquida de Alta Pressão , Cinética , Modelos Moleculares , Inibidores de Proteases/farmacologia , Conformação Proteica , Coelhos , Reticulócitos/efeitos dos fármacos , Reticulócitos/fisiologia , Proteínas Inativadoras de Ribossomos/isolamento & purificação , Proteínas Inativadoras de Ribossomos Tipo 1/química , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Saponaria/química , Saporinas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A novel ribosome-inactivating protein, designated Trichosanthrip, was purified from mature seeds of Trichosanthes kirilowii Maxim by cation-exchange and gel-filtration chromatography. Trichosanthrip migrated as a single band in SDS-PAGE, with an apparent molecular mass of approximately 13kDa. The molecular mass of Trichosanthrip was 10,964.617Da as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Trichosanthrip showed N-glycosidase activity on 28 S rRNA and strongly inhibited cell-free protein synthesis, with an IC(50) of 1.6ng/ml. Liquid chromatography-tandem mass spectrometry showed that Trichosanthrip was a novel protein with similar sequence to other proteins present in members of the Cucurbitaceae.
Assuntos
Proteínas de Plantas/química , Proteínas Inativadoras de Ribossomos Tipo 1/química , Trichosanthes/química , Cromatografia Líquida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , RNA Ribossômico 28S/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Sementes/química , Espectrometria de Massas em TandemRESUMO
A novel type 1 ribosome-inactivating protein (RIP) designated cucurmosin was isolated from the sarcocarp of Cucurbita moschata (pumpkin). Besides rRNA N-glycosidase activity, cucurmosin exhibits strong cytotoxicities to three cancer cell lines of both human and murine origins, but low toxicity to normal cells. Plant genomic DNA extracted from the tender leaves was amplified by PCR between primers based on the N-terminal sequence and X-ray sequence of the C-terminal. The complete mature protein sequence was obtained from N-terminal protein sequencing and partial DNA sequencing, confirmed by high resolution crystal structure analysis. The crystal structure of cucurmosin has been determined at 1.04A, a resolution that has never been achieved before for any RIP. The structure contains two domains: a large N-terminal domain composed of seven alpha-helices and eight beta-strands, and a smaller C-terminal domain consisting of three alpha-helices and two beta-strands. The high resolution structure established a glycosylation pattern of GlcNAc(2)Man(3)Xyl. Asn225 was identified as a glycosylation site. Residues Tyr70, Tyr109, Glu158 and Arg161 define the active site of cucurmosin as an RNA N-glycosidase. The structural basis of cytotoxicity difference between cucurmosin and trichosanthin is discussed.
Assuntos
Antineoplásicos/química , Cucurbita/química , Proteínas de Plantas/química , Proteínas Inativadoras de Ribossomos Tipo 1/química , Animais , Antineoplásicos/isolamento & purificação , Domínio Catalítico , Linhagem Celular Tumoral , Cristalografia por Raios X , DNA de Plantas , Genoma de Planta , Glicosilação , Humanos , Folhas de Planta/química , Proteínas de Plantas/isolamento & purificação , Estrutura Secundária de Proteína , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Análise de SequênciaRESUMO
Saporin, a ribosome inactivating protein is widely used for immunotoxin construction. Here we describe a mutation of saporin (sap)-3 DNA by introducing a cysteine residue, followed by protein expression and purification by ion exchange chromatography. The purified Cys255sap-3, sap-3 isomer and commercially purchased saporin, were tested for toxicity using assays measuring inhibition for protein synthesis. The IC(50) values showed that the toxicity of the Cys255sap-3 is equivalent to the sap-3 isomer and commercial saporin. Reactivity of Cys255sap-3 was confirmed by labeling with a thio-specific fluorescent probe as well as conjugation with a nonspecific mouse IgG. We have found that a single cysteine within saporin provides a method for antibody conjugation that ensures a uniform and reproducible modification of a saporin variant retaining high activity.
Assuntos
Cisteína/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Proteínas Inativadoras de Ribossomos Tipo 1/biossíntese , Proteínas Inativadoras de Ribossomos Tipo 1/genética , Animais , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Humanos , Imunoglobulina G/imunologia , Imunotoxinas/farmacologia , Maleimidas/química , Camundongos , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Coelhos , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Ribossomos/efeitos dos fármacos , SaporinasRESUMO
PD-L4, a type 1 ribosome inactivating protein from Phytolacca dioica leaves, has been successfully crystallized using vapour diffusion methods and PEG 4000 as a precipitant agent. In addition, crystals of a PD-L4 mutant, which has been recently observed to have a lower polynucleotide-adenosine glycosidase activity on DNA, rRNA and poly (A) substrates, have been obtained. To gather information on PD-L4 reaction mechanism both forms have been co-crystallized with adenine, the major product of their catalytic reaction. Diffraction patterns extend to atomic resolution and crystals belong to the orthorhombic P2(1)2(1)2(1) space group, with one molecule in the asymmetric unit. Structure determination has been achieved using molecular replacement; preliminary electron density maps have clearly given evidence of adenine binding.
Assuntos
Phytolacca/química , Folhas de Planta/química , Proteínas de Plantas/química , Proteínas Inativadoras de Ribossomos Tipo 1/química , Ribossomos/metabolismo , Cristalização , Proteínas de Plantas/isolamento & purificação , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Difração de Raios XRESUMO
The complete amino acid sequence of lychnin, a type 1 ribosome-inactivating protein (RIP) isolated from Lychnis chalcedonica seeds, has been determined by automated Edman degradation and ESI-QTOF mass spectrometry. Lychnin consists of 234 amino acid residues with a molecular mass of 26 131.14 Da. All amino acid residues involved in the formation of the RIP active site (Tyr69, Tyr119, Glu170, Arg173 and Trp203) are fully conserved. Furthermore, a fast MALDI-TOF experiment showed that two out of three cysteinyl residues (Cys32 and Cys115) form a disulfide bridge, while Cys214 is in the thiol form, which makes it suitable for linking carrier molecules to generate immunotoxins and other conjugates.