RESUMO
Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that irreversibly inhibit protein synthesis and consequently cause cell death. Recently, an RIP called ledodin has been found in shiitake; it is cytotoxic, strongly inhibits protein synthesis, and shows rRNA N-glycosylase activity. In this work, we isolated and characterized a 50 kDa cytotoxic protein from shiitake that we named edodin. Edodin inhibits protein synthesis in a mammalian cell-free system, but not in insect-, yeast-, and bacteria-derived systems. It exhibits rRNA N-glycosylase and DNA-nicking activities, which relate it to plant RIPs. It was also shown to be toxic to HeLa and COLO 320 cells. Its structure is not related to other RIPs found in plants, bacteria, or fungi, but, instead, it presents the characteristic structure of the fold type I of pyridoxal phosphate-dependent enzymes. Homologous sequences have been found in other fungi of the class Agaricomycetes; thus, edodin could be a new type of toxin present in many fungi, some of them edible, which makes them of great interest in health, both for their involvement in food safety and for their potential biomedical and biotechnological applications.
Assuntos
Ribossomos , Cogumelos Shiitake , Humanos , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Cogumelos Shiitake/química , Células HeLa , Animais , Micotoxinas/toxicidade , Micotoxinas/química , Proteínas Inativadoras de Ribossomos/química , Proteínas Inativadoras de Ribossomos/farmacologia , Proteínas Fúngicas/química , Proteínas Fúngicas/toxicidade , Proteínas Fúngicas/farmacologia , Proteínas Fúngicas/metabolismo , Linhagem Celular TumoralRESUMO
The control of crop diseases caused by fungi remains a major problem and there is a need to find effective fungicides that are environmentally friendly. Plants are an excellent source for this purpose because they have developed defense mechanisms to cope with fungal infections. Among the plant proteins that play a role in defense are ribosome-inactivating proteins (RIPs), enzymes obtained mainly from angiosperms that, in addition to inactivating ribosomes, have been studied as antiviral, fungicidal, and insecticidal proteins. In this review, we summarize and discuss the potential use of RIPs (and other proteins with similar activity) as antifungal agents, with special emphasis on RIP/fungus specificity, possible mechanisms of antifungal action, and the use of RIP genes to obtain fungus-resistant transgenic plants. It also highlights the fact that these proteins also have antiviral and insecticidal activity, which makes them very versatile tools for crop protection.
Assuntos
Antifúngicos , Proteínas Inativadoras de Ribossomos , Proteínas Inativadoras de Ribossomos/farmacologia , Antifúngicos/farmacologia , Proteínas de Plantas/farmacologia , Proteínas de Plantas/genética , Fungos/efeitos dos fármacos , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Plantas Geneticamente Modificadas , Animais , Fungicidas Industriais/farmacologiaRESUMO
The continuing emergence of SARS-CoV-2 variants has highlighted the need to identify additional points for viral inhibition. Ribosome inactivating proteins (RIPs), such as MAP30 and Momordin which are derived from bitter melon (Momordica charantia), have been found to inhibit a broad range of viruses. MAP30 has been shown to potently inhibit HIV-1 with minimal cytotoxicity. Here we show that MAP30 and Momordin potently inhibit SARS-CoV-2 replication in A549 human lung cells (IC50 ~ 0.2 µM) with little concomitant cytotoxicity (CC50 ~ 2 µM). Both viral inhibition and cytotoxicity remain unaltered by appending a C-terminal Tat cell-penetration peptide to either protein. Mutation of tyrosine 70, a key residue in the active site of MAP30, to alanine completely abrogates both viral inhibition and cytotoxicity, indicating the involvement of its RNA N-glycosylase activity. Mutation of lysine 171 and lysine 215, residues corresponding to those in Ricin which when mutated prevented ribosome binding and inactivation, to alanine in MAP30 decreased cytotoxicity (CC50 ~ 10 µM) but also the viral inhibition (IC50 ~ 1 µM). Unlike with HIV-1, neither Dexamethasone nor Indomethacin exhibited synergy with MAP30 in the inhibition of SARS-CoV-2. From a structural comparison of the two proteins, one can explain their similar activities despite differences in both their active-sites and ribosome-binding regions. We also note points on the viral genome for potential inhibition by these proteins.
Assuntos
COVID-19 , Soropositividade para HIV , HIV-1 , Momordica charantia , Humanos , Lisina , SARS-CoV-2 , Alanina , Proteínas Inativadoras de Ribossomos/farmacologia , Ribossomos , Tratamento Farmacológico da COVID-19RESUMO
Sugar-stabilised nanomaterials have received a lot of attention in cancer therapy in recent years due to their pronounced application as specific targeting agents and maximizing their therapeutic potential while bypassing off-target effects. Lectins, the carbohydrate-binding proteins, are capable of binding to receptors present on the target cell/tissue and interact with transformed glycans better than normal cells. Besides some of the lectins exhibit anticancer activity. Conjugating sugar-stabilised NPs with lectins there for is expected to multiply the potential for the early diagnosis of cancer cells and the specific release of drugs into the tumor site. Because of the prospective applications of lectin-sugar-stabilised nanoparticle conjugates, it is important to understand their molecular interaction and physicochemical properties. Momordica charantia Seed Lectin (MCL) is a type II RIP and has been known as an anti-tumor agent. Investigation of the interaction between sugar-stabilised silver nanoparticles and MCL has been performed by fluorescence spectroscopy to explore the possibility of creating an effective biocompatible drug delivery system against cancer cells. In this regard interaction between lectin and NPs should be well-preserved, while recognizing the specific cell surface sugar. Therefore experiments were carried out in the presence and absence of specific sugar galactose. Protein intrinsic fluorescence emission is quenched at ~ 20% at saturation during the interaction without any significant shift in fluorescence emission maximum. Binding experiments reveal a good affinity. Tetrameric MCL binds to a single nanoparticle. Stern-Volmer analysis of the quenching data suggests that the interaction is via static quenching leading to complex formation. Hemagglutination experiments together with interaction studies in the presence of specific sugar show that the sugar-binding site of the lectin is distinct from the nanoparticle-binding site and cell recognition is very much intact even after binding to AgNPs. Our results propose the possibility of developing MCL-silver nanoparticle conjugate with high stability and multiple properties in the diagnosis and treatment of cancer.
Assuntos
Nanopartículas Metálicas , Momordica charantia , Lectinas/metabolismo , Açúcares/metabolismo , Momordica charantia/química , Momordica charantia/metabolismo , Prata/análise , Prata/metabolismo , Carboidratos/análise , Sementes/química , Proteínas Inativadoras de Ribossomos/farmacologia , Proteínas Inativadoras de Ribossomos/análise , Proteínas Inativadoras de Ribossomos/metabolismo , Lectinas de Plantas/farmacologia , Lectinas de Plantas/químicaRESUMO
The ribosome inactivating proteins (RIPs) efficiently decrease the microbial infections in plants. Momordica charantia MAP30 is a type I RIP that has not been investigated against plant viruses or bacteriophages. To evaluate of these activities, the recombinant MAP30 (rMAP30) was produced in the hairy roots of Nicotiana tabacum. Inoculation of 3 µg of transgenic total protein or 0.6 µg of rMAP30 against 0.1 µg of TMV reduced the leaf necrotic spots to 78.23% and 82.72%, respectively. The treatment of 0.1 µg of CMV with rMAP30 (0.6 µg) showed the reduction in the leaf necrotic spots to 85.8%. While the infection was increased after rMAP30 dilution. In the time interval assays, the leaves were first inoculated with 1 µg of rMAP30 or 0.1 µg of purified TMV or CMV agent for 6 h, then virus or protein was applied in order. This led the spot reduction to 35.22% and 67% for TMV, and 38.61% and 55.31% for CMV, respectively. In both the pre- and co-treatments of 1:10 or 1:20 diluted bacteriophage with 15 µg of transgenic total protein, the number and diameter of the plaques were reduced. The results showed that the highest inhibitory effect was observed in the pre-treatment assay of bacteriophage with transgenic total protein for 24 h. The decrease in the growth of bacteriophage caused more growth pattern of Escherichia coli. The results confirm that rMAP30 shows antibacterial activity against Streptococcus aureus and E. coli, antifungal activity against Candida albicans, and antiviral activity against CMV and TMV. Moreover, rMAP30 exhibits anti-phage activity for the first time. According to our findings, rMAP30 might be a valuable preservative agent in foods and beverages in the food industry as well as an antiviral and antimicrobial mixture in agriculture.
Assuntos
Bacteriófagos , Infecções por Citomegalovirus , Vírus de Plantas , Humanos , Proteínas Inativadoras de Ribossomos Tipo 2/metabolismo , Saporinas/metabolismo , Escherichia coli/metabolismo , Proteínas Inativadoras de Ribossomos/farmacologia , Antivirais/farmacologia , Proteínas de Plantas/metabolismoRESUMO
Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that catalyze the removal of a specific adenine located in the sarcin-ricin loop of the large ribosomal RNA, which leads to the irreversible inhibition of protein synthesis and, consequently, cell death. The case of elderberry (Sambucus nigra L.) is unique, since more than 20 RIPs and related lectins have been isolated and characterized from the flowers, seeds, fruits, and bark of this plant. However, these kinds of proteins have never been isolated from elderberry leaves. In this work, we have purified RIPs and lectins from the leaves of this shrub, studying their main physicochemical characteristics, sequences, and biological properties. In elderberry leaves, we found one type 2 RIP and two related lectins that are specific for galactose, four type 2 RIPs that fail to agglutinate erythrocytes, and one type 1 RIP. Several of these proteins are homologous to others found elsewhere in the plant. The diversity of RIPs and lectins in the different elderberry tissues, and the different biological activities of these proteins, which have a high degree of homology with each other, constitute an excellent source of proteins that are of great interest in diagnostics, experimental therapy, and agriculture.
Assuntos
Ricina , Sambucus nigra , Sambucus , Adenina , Sequência de Aminoácidos , Galactose , N-Glicosil Hidrolases/genética , Folhas de Planta/metabolismo , Lectinas de Plantas/farmacologia , Proteínas de Plantas/genética , Plantas/metabolismo , RNA Ribossômico , Proteínas Inativadoras de Ribossomos/metabolismo , Proteínas Inativadoras de Ribossomos/farmacologia , Ribossomos/metabolismo , Ricina/metabolismo , Sambucus nigra/genética , Sambucus nigra/metabolismoRESUMO
Ribosome-inactivating proteins (RIPs) are known as RNA N-glycosylases. They depurinate the major rRNA, damaging ribosomes and inhibiting protein synthesis. Here, new single-chain (type-1) RIPs named sodins were isolated from the seeds (five proteins), edible leaves (one protein) and roots (one protein) of Salsola soda L. Sodins are able to release Endo's fragment when incubated with rabbit and yeast ribosomes and inhibit protein synthesis in cell-free systems (IC50 = 4.83-79.31 pM). In addition, sodin 5, the major form isolated from seeds, as well as sodin eL and sodin R, isolated from edible leaves and roots, respectively, display polynucleotide:adenosine glycosylase activity and are cytotoxic towards the Hela and COLO 320 cell lines (IC50 = 0.41-1200 nM), inducing apoptosis. The further characterization of sodin 5 reveals that this enzyme shows a secondary structure similar to other type-1 RIPs and a higher melting temperature (Tm = 76.03 ± 0.30 °C) and is non-glycosylated, as other sodins are. Finally, we proved that sodin 5 possesses antifungal activity against Penicillium digitatum.
Assuntos
Salsola , Sequência de Aminoácidos , Animais , Células HeLa , Humanos , N-Glicosil Hidrolases/química , Proteínas de Plantas/química , Coelhos , Proteínas Inativadoras de Ribossomos/metabolismo , Proteínas Inativadoras de Ribossomos/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1 , Ribossomos/metabolismo , Salsola/metabolismoRESUMO
Ribosome-inactivating proteins (RIPs) are found in several edible plants and are well characterized. Many studies highlight their use in cancer therapy, alone or as immunoconjugates, linked to monoclonal antibodies directed against target cancer cells. In this context, we investigate the cytotoxicity of quinoin, a novel type 1 RIP from quinoa seeds, on human continuous and primary glioblastoma cell lines. The cytotoxic effect of quinoin was assayed on human continuous glioblastoma U87Mg cells. Moreover, considering that common conventional glioblastoma multiforme (GBM) cell lines are genetically different from the tumors from which they derive, the cytotoxicity of quinoin was subsequently tested towards primary cells NULU and ZAR (two cell lines established from patients' gliomas), also in combination with the chemotherapeutic agent temozolomide (TMZ), currently used in glioblastoma treatment. The present study demonstrated that quinoin (2.5 and 5.0 nM) strongly reduced glioblastoma cells' growth. The mechanisms responsible for the inhibitory action of quinoin are different in the tested primary cell lines, reproducing the heterogeneous response of glioblastoma cells. Interestingly, primary cells treated with quinoin in combination with TMZ were more sensitive to the treatment. Overall, our data highlight that quinoin could represent a novel tool for glioblastoma therapy and a possible adjuvant for the treatment of the disease in combination with TMZ, alone or as possible immunoconjugates/nanoconstructs.
Assuntos
Glioblastoma/tratamento farmacológico , Proteínas de Plantas/farmacologia , Proteínas Inativadoras de Ribossomos/farmacologia , Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chenopodium quinoa/enzimologia , Humanos , Sementes/enzimologia , Temozolomida/farmacologiaRESUMO
Ribosome-inactivating proteins (RIPs) are a group of proteins exhibiting N-glycosidase activity leading to an inactivation of protein synthesis. Thirteen predicted Jatropha curcas RIP sequences could be grouped into RIP types 1 or 2. The expression of the RIP genes was detected in seed kernels, seed coats, and leaves. The full-length cDNA of two RIP genes (26SK and 34.7(A)SK) were cloned and studied. The 34.7(A)SK protein was successfully expressed in the host cells while it was difficult to produce even only a small amount of the 26SK protein. Therefore, the crude proteins were used from E. coli expressing 26SK and 34.7(A)SK constructs and they showed RIP activity. Only the cell lysate from 26SK could inhibit the growth of E. coli. In addition, the crude protein extracted from 26SK expressing cells displayed the effect on the growth of MDA-MB-231, a human breast cancer cell line. Based on in silico analysis, all 13 J. curcas RIPs contained RNA and ribosomal P2 stalk protein binding sites; however, the C-terminal region of the P2 stalk binding site was lacking in the 26SK structure. In addition, an amphipathic distribution between positive and negative potential was observed only in the 26SK protein, similar to that found in the anti-microbial peptide. These findings suggested that this 26SK protein structure might have contributed to its toxicity, suggesting potential uses against pathogenic bacteria in the future.
Assuntos
Escherichia coli/metabolismo , Jatropha/química , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Inativadoras de Ribossomos , Humanos , Proteínas Inativadoras de Ribossomos/química , Proteínas Inativadoras de Ribossomos/farmacologiaRESUMO
Ribosome-inactivating proteins (RIPs) are rRNA N-glycosylases from plants (EC 3.2.2.22) that inactivate ribosomes thus inhibiting protein synthesis. The antiviral properties of RIPs have been investigated for more than four decades. However, interest in these proteins is rising due to the emergence of infectious diseases caused by new viruses and the difficulty in treating viral infections. On the other hand, there is a growing need to control crop diseases without resorting to the use of phytosanitary products which are very harmful to the environment and in this respect, RIPs have been shown as a promising tool that can be used to obtain transgenic plants resistant to viruses. The way in which RIPs exert their antiviral effect continues to be the subject of intense research and several mechanisms of action have been proposed. The purpose of this review is to examine the research studies that deal with this matter, placing special emphasis on the most recent findings.
Assuntos
Antivirais/farmacologia , Controle Biológico de Vetores , Doenças das Plantas/prevenção & controle , Plantas Geneticamente Modificadas/enzimologia , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Inativadoras de Ribossomos/farmacologia , Toxinas Biológicas/farmacologia , Viroses/tratamento farmacológico , Vírus/efeitos dos fármacos , Animais , Antivirais/isolamento & purificação , Humanos , Doenças das Plantas/genética , Doenças das Plantas/virologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/virologia , Inibidores da Síntese de Proteínas/isolamento & purificação , Proteínas Inativadoras de Ribossomos/isolamento & purificação , Toxinas Biológicas/isolamento & purificação , Viroses/metabolismo , Viroses/virologia , Vírus/metabolismo , Vírus/patogenicidadeRESUMO
Gene-regulatory networks reconstruction has become a very popular approach in applied biology to infer and dissect functional interactions of Transcription Factors (TFs) driving a defined phenotypic state, termed as Master Regulators (MRs). In the present work, cutting-edge bioinformatic methods were applied to re-analyze experimental data on leukemia cells (human myelogenous leukemia cell line THP-1 and acute myeloid leukemia MOLM-13 cells) treated for 6 h with two different Ribosome-Inactivating Proteins (RIPs), namely Shiga toxin type 1 (400 ng/mL) produced by Escherichia coli strains and the plant toxin stenodactylin (60 ng/mL), purified from the caudex of Adenia stenodactyla Harms. This analysis allowed us to identify the common early transcriptional response to 28S rRNA damage based on gene-regulatory network inference and Master Regulator Analysis (MRA). Both toxins induce a common response at 6 h which involves inflammatory mediators triggered by AP-1 family transcriptional factors and ATF3 in leukemia cells. We describe for the first time the involvement of MAFF, KLF2 and KLF6 in regulating RIP-induced apoptotic cell death, while receptor-mediated downstream signaling through ANXA1 and TLR4 is suggested for both toxins.
Assuntos
Redes Reguladoras de Genes/efeitos dos fármacos , Leucemia/metabolismo , Proteínas Inativadoras de Ribossomos/farmacologia , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Lectinas/farmacologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , N-Glicosil Hidrolases/farmacologia , Toxina Shiga I/farmacologia , Fatores de Transcrição/metabolismoRESUMO
Glycoforms are otherwise identical proteins with different glycosylation. A lectin, Sambucus sieboldiana agglutinin (SSA), specifically binds glycoforms having α2,6-sialyl residues. The binding is found to inhibit antigen-antibody reaction; e.g., SSA inhibits anti-transferrin antibody binding to α2,6-sialylated transferrin (Tf) (SSA inhibition). SSA inhibition is not observed with other Tf glycoforms, indicating that the inhibition is glycoform-specific. Here we describe the application of SSA inhibition to ELISA as a specific assay for quantifying α2,6-sialylated Tf.
Assuntos
Lectinas de Plantas/farmacologia , Proteínas Inativadoras de Ribossomos/farmacologia , Sambucus/metabolismo , Transferrina/análise , Transferrina/química , Anticorpos/metabolismo , Reações Antígeno-Anticorpo/efeitos dos fármacos , Análise Química do Sangue , Ensaio de Imunoadsorção Enzimática , Glicosilação , Humanos , Ácido N-Acetilneuramínico/metabolismo , Transferrina/imunologiaRESUMO
Antibodies are useful for localizing glycoprotein antigens in histochemistry, but they do not differentiate glycoforms in tissue sections because conventional antibodies recognize only protein epitopes rather than glycans. Glycan epitopes are recognized by lectins, which are found, occasionally, to inhibit antigen-antibody reaction in a glycoform-specific manner (lectin inhibition). Here we describe the application of lectin inhibition to immunohistochemistry for visualizing a glycoform in a tissue section.
Assuntos
Glicoproteínas/análise , Fígado/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Lectinas de Plantas/farmacologia , Proteínas Inativadoras de Ribossomos/farmacologia , Animais , Reações Antígeno-Anticorpo/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Glicoproteínas/química , Humanos , Imuno-Histoquímica , Micrococcaceae , Transferrina/análise , Transferrina/químicaRESUMO
Ribosome-inactivating proteins (RIPs) are N-glycosidases, which depurinate a specific adenine residue in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. This loop is important for anchoring elongation factor (EF-G for prokaryote or eEF2 for eukaryote) in mRNA translocation. Translation is inhibited after the attack. RIPs therefore may have been applied for anti-cancer, and anti-virus and other therapeutic applications. The main obstacles of treatment with RIPs include short plasma half-life, non-selective cytotoxicity and antigenicity. This review focuses on the strategies used to improve the pharmacological properties of RIPs on human immunodeficiency virus (HIV) and cancers. Coupling with polyethylene glycol (PEG) increases plasma time and reduces antigenicity. RIPs conjugated with antibodies to form immunotoxins increase the selective toxicity to target cells. The prospects for future development on the engineering of RIPs for improving their pharmacological properties are also discussed.
Assuntos
Proteínas Inativadoras de Ribossomos , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Dextranos/química , Dextranos/farmacologia , Dextranos/uso terapêutico , Humanos , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Polietilenoglicóis/uso terapêutico , Engenharia de Proteínas , Proteínas Inativadoras de Ribossomos/química , Proteínas Inativadoras de Ribossomos/farmacologia , Proteínas Inativadoras de Ribossomos/uso terapêuticoRESUMO
Ribosome-inactivating proteins (RIPs) consist of three varieties. Type 1 RIPs are single-chained and approximately 30-kDa in molecular weight. Type 2 RIPs are double-chained and composed of a type 1 RIP chain and a lectin chain. Type III RIPs, such as maize b-32 barley and JIP60 which are produced as single-domain proenzymes, possess an N-terminal domain corresponding to the A domain of RIPs and fused to a C-terminal domain. In addition to the aforementioned three types of RIPs originating from flowering plants, there are recently discovered proteins and peptides with ribosome-inactivating and protein synthesis inhibitory activities but which are endowed with characteristics such as molecular weights distinctive from those of the regular RIPs. These new/unusual RIPs discussed in the present review encompass metazoan RIPs from Anopheles and Culex mosquitos, antimicrobial peptides derived from RIP of the pokeweed Phytolacca dioica, maize RIP (a type III RIP derived from a precursor form), RIPs from the garden pea and the kelp. In addition, RIPs with a molecular weight smaller than those of regular type 1 RIPs are produced by plants in the Cucurbitaceae family including the bitter gourd, bottle gourd, sponge gourd, ridge gourd, wax gourd, hairy gourd, pumpkin, and Chinese cucumber. A small type II RIP from camphor tree (Cinnamomum camphora) seeds and a snake gourd type II RIP with its catalytic chain cleaved into two have been reported. RIPs produced from mushrooms including the golden needle mushroom, king tuber mushroom, straw mushroom, and puffball mushroom are also discussed in addition to a type II RIP from the mushroom Polyporus umbellatus. Bacterial (Spiroplasma) RIPs associated with the fruitfly, Shiga toxin, and Streptomyces coelicolor RIP are also dealt with. The aforementioned proteins display a diversity of molecular weights, amino acid sequences, and mechanisms of action. Some of them are endowed with exploitable antipathogenic activities.
Assuntos
Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Inativadoras de Ribossomos/metabolismo , Sequência de Aminoácidos , Animais , Culicidae/química , Proteínas de Insetos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Inativadoras de Ribossomos/classificação , Proteínas Inativadoras de Ribossomos/farmacologia , Sementes/químicaRESUMO
Alpha-momorcharin (α-MMC), a member of the ribosome-inactivating protein (RIP) family, has been found in the seeds of Momordica charantia (bitter melon). α-MMC contributes a number of pharmacological activities; however, its inflammatory properties have not been well studied. Here, we aim to determine the inflammatory responses induced by recombinant α-MMC and identify the underlying mechanisms using cell culture and animal models. Recombinant α-MMC was generated in Rosetta™(DE3)pLysS and purified by the way of nitrilotriacetic acid (NTA) chromatography. Treatment of recombinant α-MMC at 40 µg/mL exerted sub-lethal cytotoxic effect on THP-1 monocytic cells. Transcriptional profiling revealed that various genes coding for cytokines and other proinflammatory proteins were upregulated upon recombinant α-MMC treatment in THP-1 cells, including MCP-1, IL-8, IL-1ß, and TNF-α. Recombinant α-MMC was shown to activate IKK/NF-κB and JNK pathways and the α-MMC-induced inflammatory gene expression could be blocked by IKKß and JNK inhibitors. Furthermore, murine inflammatory models further demonstrated that α-MMC induced inflammatory responses in vivo. We conclude that α-MMC stimulates inflammatory responses in human monocytes by activating of IKK/NF-κB and JNK pathways, raising the possibility that consumption of α-MMC-containing food may lead to inflammatory-related diseases.
Assuntos
Inflamação/induzido quimicamente , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Momordica charantia/química , NF-kappa B/efeitos dos fármacos , Proteínas Inativadoras de Ribossomos/química , Animais , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocinas/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Análise em Microsséries , Plantas Comestíveis , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Proteínas Inativadoras de Ribossomos/farmacologiaRESUMO
Background and aim: Alpha-momorcharin (α-MMC) is a type I ribosome-inactivating protein (RIP) that is purified from Momordica charantia. Despite its strong antitumor activities, α-MMC exerts the undesirable immunotoxicity effects of hypersensitivity or immunosuppression. Since α-MMC is a plant protein, its application in vivo can easily induce hypersensitivity, but its immunosuppressive mechanism is still unclear. Materials and methods: The toxicity of α-MMC to peripheral blood cells and the cytokine expression in peripheral blood mononuclear cells (PBMCs) and spleen immune cells were measured in rats. For further confirmation, experiments were performed in vitro with the mononuclear cell line THP-1, B lymphocyte cell line WIL2-S and T lymphocyte cell line Jurkat. Results: High doses of α-MMC (3.0 mg/kg) resulted in weight loss in rats, a decreased percentage of monocytes, and increased percentages of eosinophils and basophils. Both high-dose and low-dose (1.0 mg/kg) α-MMC inhibited cytokine expression in PBMCs and increased cytokine expression in spleen T cells. In in vitro, α-MMC mainly acted on THP-1 cells, with effects including high dose-induced apoptosis and low dose-induced regulation of inhibitory cytokine expression. Conclusions: The action of α-MMC on immune cells mainly affects monocytes, thereby eliciting its immunosuppressive effect. Its mode of action is to guide functional immunosuppressive regulation at low doses and induce apoptosis at high doses. As the monocytes would be recruited into tumor tissues and are polarized into tumor-associated macrophages, the selective cytotoxicity and cytokine release regulation of α-MMC in monocytes may be an important mechanism of its antitumor effects.
Assuntos
Apoptose/efeitos dos fármacos , Citocinas/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Monócitos/imunologia , Proteínas Inativadoras de Ribossomos/farmacologia , Animais , Apoptose/imunologia , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Células Jurkat , Monócitos/patologia , Ratos , Ratos Sprague-Dawley , Células THP-1RESUMO
It is reasonable to consider that Type I-ribosomal inactivation proteins (RIP) retain some specific affinity to harmful pathogens to complete the role as a bio-defense relating protein. In the present studies, it was shown that two Type I-RIPs, α- and ß-momorcharins, maintained the abilities to bind with N-acetylglucosamine (NAG) to change the conformation around the active sites and to regulate their N-glycosidase activities. By the binding of NAG, the freedom of internal motion of Trp192 in α-momorcharin was increased 1.5 times near the active site and, on the other hand, the corresponding motion of Trp190 was limited 50% in ß-momorcharin. The results in the fluorescence resonance excitation energy transfer experiments demonstrated that Trp-190 of ß-momorcharin was kept away from Tyr-70 but Trp192 contrarily approached closer to the nearest neighboring Tyr residue consisting of the active center of α-momorcharin by the binding with NAG. These conformational changes near the active site close correlated with promotion and/or suppression of the N-glucosidase activities of ß- and α-momorcharins.
Assuntos
Momordica charantia/química , N-Glicosil Hidrolases/antagonistas & inibidores , Proteínas Inativadoras de Ribossomos/farmacologia , Sementes/química , Domínio Catalítico/efeitos dos fármacos , Ligantes , Modelos Moleculares , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/metabolismo , Conformação Proteica , Proteínas Inativadoras de Ribossomos/químicaRESUMO
Plant ribosome-inactivating proteins (RIPs) are a family of toxins that inhibit protein synthesis. In this study, we have isolated a novel type 2 ribosome-inactivating protein (RIP) present in seeds of the Abrus fruticulosus, named of fruticulosin. Fruticulosin, shows characteristics common to other type 2 RIPs, as specificity by galactosides (d-galactose, N-acetyl-d-galactosamine, and d-lactose), mass of approximately 60â¯kDa and presence of the of disulfide bonds. The N-terminal amino acid sequence (26 residues) of A-chain fruticulosin, determined by Edman degradation, revealed high similarity of the A-chain with those of other type 2 RIPs. The secondary structure of fruticulosin was analysed by circular dichroism, which showed that fruticulosin contains α-helices (22.3%), ß-sheets (43.5%), and random coils and corners (34.2%). Furthermore, fruticulosin showed high toxicity in Artemia sp. (3.12⯵g/mL), inhibited in vitro protein synthesis by a cell-free system and showed RNA N-glycosidase activity. Fruticulosin presented biological activities such as agglutination and antileishmanial activity on promastigote forms of Leishmania major.
Assuntos
Abrus/química , Proteínas de Plantas/farmacologia , Proteínas Inativadoras de Ribossomos/farmacologia , Tripanossomicidas/farmacologia , Sequência de Aminoácidos , Animais , Artemia/efeitos dos fármacos , Hemaglutininas/química , Hemaglutininas/isolamento & purificação , Hemaglutininas/farmacologia , Hemaglutininas/toxicidade , Leishmania major/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Parasitária , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/toxicidade , Inibidores da Síntese de Proteínas/química , Inibidores da Síntese de Proteínas/isolamento & purificação , Inibidores da Síntese de Proteínas/farmacologia , Inibidores da Síntese de Proteínas/toxicidade , Coelhos , Proteínas Inativadoras de Ribossomos/química , Proteínas Inativadoras de Ribossomos/isolamento & purificação , Proteínas Inativadoras de Ribossomos/toxicidade , Sementes/química , Homologia de Sequência de Aminoácidos , Tripanossomicidas/química , Tripanossomicidas/isolamento & purificação , Tripanossomicidas/toxicidadeRESUMO
Bougainvillea (Bougainvillea spectabilis Willd.) is a plant widely used in folk medicine and many extracts from different tissues of this plant have been employed against several pathologies. The observation that leaf extracts of Bougainvillea possess antiviral properties led to the purification and characterization of a protein, named bouganin, which exhibits typical characteristics of type 1 ribosome-inactivating proteins (RIPs). Beyond that, bouganin has some peculiarities, such as a higher activity on DNA with respect to ribosomal RNA, low systemic toxicity, and immunological properties quite different than other RIPs. The sequencing of bouganin and the knowledge of its three-dimensional structure allowed to obtain a not immunogenic mutant of bouganin. These features make bouganin a very attractive tool as a component of immunotoxins (ITs), chimeric proteins obtained by linking a toxin to a carrier molecule. Bouganin-containing ITs showed very promising results in the experimental treatment of both hematological and solid tumors, and one bouganin-containing IT has entered Phase I clinical trial. In this review, we summarize the milestones of the research on bouganin such as bouganin chemico-physical characteristics, the structural properties and de-immunization studies. In addition, the in vitro and in vivo results obtained with bouganin-containing ITs are summarized.