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1.
Biosensors (Basel) ; 14(1)2024 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-38248411

RESUMO

Pap smear screening is a widespread technique used to detect premalignant lesions of cervical cancer (CC); however, it lacks sensitivity, leading to identifying biomarkers that improve early diagnosis sensitivity. A characteristic of cancer is the aberrant sialylation that involves the abnormal expression of α2,6 sialic acid, a specific carbohydrate linked to glycoproteins and glycolipids on the cell surface, which has been reported in premalignant CC lesions. This work aimed to develop a method to differentiate CC cell lines and primary fibroblasts using a novel lectin-based biosensor to detect α2,6 sialic acid based on attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and chemometric. The biosensor was developed by conjugating gold nanoparticles (AuNPs) with 5 µg of Sambucus nigra (SNA) lectin as the biorecognition element. Sialic acid detection was associated with the signal amplification in the 1500-1350 cm-1 region observed by the surface-enhanced infrared absorption spectroscopy (SEIRA) effect from ATR-FTIR results. This region was further analyzed for the clustering of samples by applying principal component analysis (PCA) and confidence ellipses at a 95% interval. This work demonstrates the feasibility of employing SNA biosensors to discriminate between tumoral and non-tumoral cells, that have the potential for the early detection of premalignant lesions of CC.


Assuntos
Nanopartículas Metálicas , Lectinas de Plantas , Proteínas Inativadoras de Ribossomos , Sambucus nigra , Neoplasias do Colo do Útero , Feminino , Humanos , Neoplasias do Colo do Útero/diagnóstico , Lectinas , Ácido N-Acetilneuramínico , Ouro , Linhagem Celular
2.
Biochim Biophys Acta Rev Cancer ; 1878(6): 189018, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37944831

RESUMO

Dysregulated protein synthesis is a hallmark of tumors. mRNA translation reprogramming contributes to tumorigenesis, which is fueled by abnormalities in ribosome formation, tRNA abundance and modification, and translation factors. Not only malignant cells but also stromal cells within tumor microenvironment can undergo transformation toward tumorigenic phenotypes during translational reprogramming. Ribosome-inactivating proteins (RIPs) have garnered interests for their ability to selectively inhibit protein synthesis and suppress tumor growth. This review summarizes the role of dysregulated translation machinery in tumor development and explores the potential of RIPs in cancer treatment.


Assuntos
Neoplasias , Proteínas Inativadoras de Ribossomos , Humanos , Proteínas Inativadoras de Ribossomos/genética , Proteínas Inativadoras de Ribossomos/uso terapêutico , Proteínas Inativadoras de Ribossomos/metabolismo , Ribossomos/genética , Biossíntese de Proteínas , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Microambiente Tumoral
3.
Int J Biol Macromol ; 248: 125929, 2023 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-37481176

RESUMO

Ribosome-inactivating proteins (RIPs) are found in bacteria, fungi, and plants, with a wide range of biological resistances such as anti-fungal, anti-viral, anti-insect, and anti-tumor. They can be roughly divided into proactive defense bacterial or fungal types and passive defense plant types. We identified 1592 RIP genes in bacteria, fungi, and plants. Approximately 88 % of the 764 bacterial RIPs were Shiga or Shiga-like toxins which were exotoxins and could rapidly enter cells to possess strong biotoxicity, and about 98 % of fungal RIPs were predicted as secreted proteins. RIPs were not detected in non-seed plants such as algae, bryophytes, and ferns. However, we found RIPs in some flowering and non-flowering seed plants. The existence of plant RIPs might be related to the structure of seeds or fruits, which might be associated with whether seeds are easy to survive and spread. The evolutionary characteristics of RIPs were different between dicotyledons and monocotyledons. In addition, we also found that RIP2 genes might emerge very early and be plant-specific. Some plant RIP1 genes might evolve from RIP2 genes. This study provides new insights into the evolution of RIPs.


Assuntos
Plantas , Proteínas Inativadoras de Ribossomos , Proteínas Inativadoras de Ribossomos/genética , Proteínas Inativadoras de Ribossomos/metabolismo , Plantas/genética , Plantas/metabolismo , Bactérias/genética , Bactérias/metabolismo , Ribossomos/metabolismo , Fungos/genética , Fungos/metabolismo , Seleção Genética , Proteínas de Plantas/química
4.
Gene ; 877: 147547, 2023 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-37286020

RESUMO

Ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that depurinate an adenine residue in the conserved alpha-sarcin/ricin loop (SRL) of rRNA, inhibiting protein synthesis. Previously, we reported the existence of these toxins in insects, whose presence is restricted to mosquitoes from the Culicinae subfamily (e.g., Aedes aegypti) and whiteflies from the Aleyrodidae family (e.g., Bemisia tabaci). Both groups of genes are derived from two independent horizontal gene transfer (HGT) events and are evolving under purifying selection. Here, we report and characterize the occurrence of a third HGT event in the Sciaroidea superfamily, which supports the recurrent acquisition of RIP genes by insects. Transcriptomic experiments, available in databases, allowed us to describe the temporal and spatial expression profiles for these foreign genes in these organisms. Furthermore, we found that RIP expression is induced after infection with pathogens and provided, for the first time, transcriptomic evidence of parasite SRL depurination. This evidence suggests a possible role of these foreign genes as immune effectors in insects.


Assuntos
Hemípteros , Ricina , Animais , Proteínas Inativadoras de Ribossomos/genética , Proteínas Inativadoras de Ribossomos/metabolismo , Transferência Genética Horizontal , Insetos/genética , Biossíntese de Proteínas , RNA Ribossômico , Ricina/química , Ricina/genética , Ricina/metabolismo , Hemípteros/genética , Hemípteros/metabolismo , Proteínas de Plantas/genética
5.
PLoS One ; 18(6): e0286370, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37384752

RESUMO

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-19
6.
Glycoconj J ; 40(2): 179-189, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36800135

RESUMO

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/química
7.
Sci Rep ; 13(1): 2091, 2023 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-36747030

RESUMO

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/metabolismo
8.
Toxins (Basel) ; 15(1)2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36668855

RESUMO

After more than 50 years of research, studies on the structure and biological activities of ribosome-inactivating proteins (RIPs) continue to provide a field of great interest within the scientific community, both for the health risks they pose and their applications in medicine and biotechnology [...].


Assuntos
Proteínas Inativadoras de Ribossomos , Ribossomos , Proteínas Inativadoras de Ribossomos/química , Ribossomos/metabolismo , Proteínas de Plantas/metabolismo
9.
IUBMB Life ; 75(2): 82-96, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36121739

RESUMO

Ribosome-inactivating proteins (RIPs) are toxic proteins with N-glycosidase activity. RIPs exert their action by removing a specific purine from 28S rRNA, thereby, irreversibly inhibiting the process of protein synthesis. RIPs can target both prokaryotic and eukaryotic cells. In bacteria, the production of RIPs aid in the process of pathogenesis whereas, in plants, the production of these toxins has been attributed to bolster defense against insects, viral, bacterial and fungal pathogens. In recent years, RIPs have been engineered to target a particular cell type, this has fueled various experiments testing the potential role of RIPs in many biomedical applications like anti-viral and anti-tumor therapies in animals as well as anti-pest agents in engineered plants. In this review, we present a comprehensive study of various RIPs, their mode of action, their significance in various fields involving plants and animals. Their potential as treatment options for plant infections and animal diseases is also discussed.


Assuntos
Plantas , Proteínas Inativadoras de Ribossomos , Animais , Proteínas Inativadoras de Ribossomos/uso terapêutico , Plantas/metabolismo , Antivirais/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Proteínas de Plantas
10.
Toxins (Basel) ; 16(1)2023 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-38276525

RESUMO

Ribosome-inactivating proteins (RIPs) are plant toxins that were identified for their ability to irreversibly damage ribosomes, thereby causing arrest of protein synthesis and induction of cell death. The RIPs purified from Adenia plants are the most potent ones. Here, we describe a novel toxic lectin from Adenia heterophylla caudex, which has been named heterophyllin. Heterophyllin shows the enzymatic and lectin properties of type 2 RIPs. Interestingly, in immunoreactivity experiments, heterophyllin poorly cross-reacts with sera against all other tested RIPs. The cytotoxic effects and death pathways triggered by heterophyllin were investigated in three human-derived cell lines: NB100, T24, and MCF7, and compared to ricin, the most known and studied type 2 RIP. Heterophyllin was able to completely abolish cell viability at nM concentration. A strong induction of apoptosis, but not necrosis, and the involvement of oxidative stress and necroptosis were observed in all the tested cell lines. Therefore, the enzymatic, immunological, and biological activities of heterophyllin make it an interesting molecule, worthy of further in-depth analysis to verify its possible pharmacological application.


Assuntos
Proteínas de Plantas , Ricina , Humanos , Proteínas de Plantas/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 2/metabolismo , Ricina/toxicidade , Ricina/metabolismo , Proteínas Inativadoras de Ribossomos/toxicidade , Proteínas Inativadoras de Ribossomos/metabolismo , Ribossomos/metabolismo , Biossíntese de Proteínas
11.
Toxins (Basel) ; 14(11)2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36356021

RESUMO

Curcin and Curcin C, both of the ribosome-inactivating proteins of Jatropha curcas, have apparent inhibitory effects on the proliferation of osteosarcoma cell line U20S. However, the inhibitory effect of the latter is 13-fold higher than that of Curcin. The mechanism responsible for the difference has not been studied. This work aimed to understand and verify whether there are differences in entry efficiency and pathway between them using specific endocytosis inhibitors, gene silencing, and labeling techniques such as fluorescein isothiocyanate (FITC) labeling. The study found that the internalization efficiency of Curcin C was twice that of Curcin for U2OS cells. More than one entering pathway was adopted by both of them. Curcin C can enter U2OS cells through clathrin-dependent endocytosis and macropinocytosis, but clathrin-dependent endocytosis was not an option for Curcin. The low-density lipoprotein receptor-related protein 1 (LRP1) was found to mediate clathrin-dependent endocytosis of Curcin C. After LRP1 silencing, there was no significant difference in the 50% inhibitory concentration (IC50) and endocytosis efficiency between Curcin and Curcin C on U2OS cells. These results indicate that LRP1-mediated endocytosis is specific to Curcin C, thus leading to higher U2OS endocytosis efficiency and cytotoxicity than Curcin.


Assuntos
Alcaloides , Jatropha , Osteossarcoma , Toxinas Biológicas , Humanos , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Jatropha/genética , Jatropha/metabolismo , Proteínas Inativadoras de Ribossomos/metabolismo , Toxinas Biológicas/metabolismo , Alcaloides/metabolismo , Clatrina/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo
12.
Toxins (Basel) ; 14(9)2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36136551

RESUMO

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/metabolismo
13.
Mol Biol Rep ; 49(12): 11503-11514, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36097128

RESUMO

BACKGROUND: Tobacco is an important economic crop, but the quality and yield have been severely impaired by bacterial wilt disease (BWD) caused by Ralstonia solanacearum. METHODS AND RESULTS: Here, we describe a transgenic approach to prevent BWD in tobacco plants. A new root-specific promoter of an NtR12 gene was successfully cloned. The NtR12 promoter drove GUS reporter gene expression to a high level in roots but to less extent in stems, and no significant expression was detected in leaves. The Ribosome-inactivating proteins (RIP) gene from Momordica charantia was also cloned, and its ability to inhibit Ralstonia solanacearum was evaluated using RIP protein produced by the prokaryotic expression system. The RIP gene was constructed downstream of the NtR12 promoter and transformed into the tobacco cultivar "Cuibi No. 1" (CB-1), resulting in many descendants. The resistance against BWD was significantly improved in transgenic tobacco lines expressing NtR12::RIP. CONCLUSION: This study confirms that the RIP gene confers resistance to BWD and the NtR12 as a new promoter for its specific expression in root and stem. Our findings pave a novel avenue for transgenic engineering to prevent the harmful impact of diseases and pests in roots and stems.


Assuntos
Ralstonia solanacearum , /metabolismo , Proteínas Inativadoras de Ribossomos/genética , Proteínas Inativadoras de Ribossomos/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Ralstonia solanacearum/genética , Ralstonia solanacearum/metabolismo , Regiões Promotoras Genéticas/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Resistência à Doença/genética
14.
Int J Mol Sci ; 23(17)2022 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-36076961

RESUMO

Plukenetia volubilis is a highly promising plant with high nutritional and economic values. In our previous studies, the expression levels of ricin encoded transcripts were the highest in the maturation stage of P. volubilis seeds. The present study investigated the transcriptome and proteome profiles of seeds at two developmental stages (Pv-1 and Pv-2) using RNA-Seq and iTRAQ technologies. A total of 53,224 unigenes and 6026 proteins were identified, with functional enrichment analyses, including GO, KEGG, and KOG annotations. At two development stages of P. volubilis seeds, 8815 unique differentially expressed genes (DEGs) and 4983 unique differentially abundant proteins (DAPs) were identified. Omics-based association analysis showed that ribosome-inactivating protein (RIP) transcripts had the highest expression and abundance levels in Pv-2, and those DEGs/DAPs of RIPs in the GO category were involved in hydrolase activity. Furthermore, 21 RIP genes and their corresponding amino acid sequences were obtained from libraries produced with transcriptome analysis. The analysis of physicochemical properties showed that 21 RIPs of P. volubilis contained ricin, the ricin_B_lectin domain, or RIP domains and could be divided into three subfamilies, with the largest number for type II RIPs. The expression patterns of 10 RIP genes indicated that they were mostly highly expressed in Pv-2 and 4 transcripts encoding ricin_B_like lectins had very low expression levels during the seed development of P. volubilis. This finding would represent valuable evidence for the safety of oil production from P. volubilis for human consumption. It is also notable that the expression level of the Unigene0030485 encoding type I RIP was the highest in roots, which would be related to the antiviral activity of RIPs. This study provides a comprehensive analysis of the physicochemical properties and expression patterns of RIPs in different organs of P. volubilis and lays a theoretical foundation for further research and utilization of RIPs in P. volubilis.


Assuntos
Proteínas Inativadoras de Ribossomos , Ricina , Humanos , Proteínas de Plantas/química , Proteoma/metabolismo , Proteínas Inativadoras de Ribossomos/genética , Ricina/química , Sementes/metabolismo , Transcriptoma
15.
ACS Chem Biol ; 17(9): 2619-2630, 2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-35969718

RESUMO

Ribosome-inactivating proteins (RIPs) are RNA:adenosine glycosidases that inactivate eukaryotic ribosomes by depurinating the sarcin-ricin loop (SRL) in 28S rRNA. The GAGA sequence at the top of the SRL or at the top of a hairpin loop is assumed to be their target motif. Saporin is a RIP widely used to develop immunotoxins for research and medical applications, but its sequence specificity has not been investigated. Here, we combine the conventional aniline cleavage assay for depurinated nucleic acids with high-throughput sequencing to study sequence-specific depurination of oligonucleotides caused by saporin. Our data reveal the sequence preference of saporin for different substrates and show that the GAGA motif is not efficiently targeted by this protein, neither in RNA nor in DNA. Instead, a preference of saporin for certain hairpin DNAs was observed. The observed sequence-specific activity of saporin may be relevant to antiviral or apoptosis-inducing effects of RIPs. The developed method could also be useful for studying the sequence specificity of depurination by other RIPs or enzymes.


Assuntos
Imunotoxinas , Ricina , Adenosina , Compostos de Anilina , Antivirais/farmacologia , DNA/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Oligonucleotídeos , Proteínas de Plantas/metabolismo , RNA/metabolismo , RNA Ribossômico 28S , Proteínas Inativadoras de Ribossomos , Proteínas Inativadoras de Ribossomos Tipo 1 , Ricina/farmacologia , Saporinas
16.
Phytochemistry ; 202: 113337, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35934106

RESUMO

Ribosome inactivating proteins (RIPs) are rRNA N-glycosylases (EC 3.2.2.22) best known for hydrolyzing an adenine base from the conserved sarcin/ricin loop of ribosomal RNA. Protein translation is inhibited by ribosome depurination; therefore, RIPs are generally considered toxic to cells. The expression of some RIPs is upregulated by biotic and abiotic stress, though the connection between RNA depurination and defense response is not well understood. Despite their prevalence in approximately one-third of flowering plant orders, our knowledge of RIPs stems primarily from biochemical analyses of individuals or genomics-scale analyses of small datasets from a limited number of species. Here, we performed an unbiased search for proteins with RIP domains and identified several-fold more RIPs than previously known - more than 800 from 120 species, many with novel associated domains and physicochemical characteristics. Based on protein domain configuration, we established 15 distinct groups, suggesting diverse functionality. Surprisingly, most of these RIPs lacked a signal peptide, indicating they may be localized to the nucleocytoplasm of cells, raising questions regarding their toxicity against conspecific ribosomes. Our phylogenetic analysis significantly extends previous models for RIP evolution in plants, predicting an original single-domain RIP that later evolved to acquire a signal peptide and different protein domains. We show that RIPs are distributed throughout 21 plant orders with many species maintaining genes for more than one RIP group. Our analyses provide the foundation for further characterization of these new RIP types, to understand how these enzymes function in plants.


Assuntos
Proteínas Inativadoras de Ribossomos , Ribossomos , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sinais Direcionadores de Proteínas/genética , RNA Ribossômico/análise , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas Inativadoras de Ribossomos/química , Proteínas Inativadoras de Ribossomos/genética , Ribossomos/química , Ribossomos/genética , Ribossomos/metabolismo
17.
Toxins (Basel) ; 14(8)2022 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-36006228

RESUMO

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/metabolismo
18.
Toxins (Basel) ; 14(7)2022 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-35878187

RESUMO

Type I ribosome-inactivating proteins (RIPs) are plant toxins that inhibit protein synthesis by exerting rRNA N-glycosylase activity (EC 3.2.2.22). Due to the lack of a cell-binding domain, type I RIPs are not target cell-specific. However once linked to antibodies, so called immunotoxins, they are promising candidates for targeted anti-cancer therapy. In this study, sapovaccarin-S1 and -S2, two newly identified type I RIP isoforms differing in only one amino acid, were isolated from the seeds of Saponaria vaccaria L. Sapovaccarin-S1 and -S2 were purified using ammonium sulfate precipitation and subsequent cation exchange chromatography. The determined molecular masses of 28,763 Da and 28,793 Da are in the mass range typical for type I RIPs and the identified amino acid sequences are homologous to known type I RIPs such as dianthin 30 and saporin-S6 (79% sequence identity each). Sapovaccarin-S1 and -S2 showed adenine-releasing activity and induced cell death in Huh-7 cells. In comparison to other type I RIPs, sapovaccarin-S1 and -S2 exhibited a higher thermostability as shown by nano-differential scanning calorimetry. These results suggest that sapovaccarin-S1 and -S2 would be optimal candidates for targeted anti-cancer therapy.


Assuntos
Saponaria , Vaccaria , N-Glicosil Hidrolases/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/farmacologia , Isoformas de Proteínas , Proteínas Inativadoras de Ribossomos/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/química , Ribossomos/metabolismo , Saponaria/química , Saponaria/metabolismo , Sementes/química
19.
Toxins (Basel) ; 14(6)2022 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-35737065

RESUMO

rRNA N-glycosylases (EC 3.2.2.22) remove a specific adenine (A4324, rat 28S rRNA) in the sarcin ricin loop (SRL) involved into ribosome interaction with elongation factors, causing the inhibition of translation, for which they are known as plant 'ribosome inactivating proteins' (RIPs). However, protein synthesis inactivation could be the result of other enzymes, which often have rRNA as the target. In this scenario, Endo's assay is the most used method to detect the enzymes that are able to hydrolyze a phosphodiester bond or cleave a single N-glycosidic bond (rRNA N-glycosylases). Indeed, the detection of a diagnostic fragment from rRNA after enzymatic action, with or without acid aniline, allows one to discriminate between the N-glycosylases or hydrolases, which release the ß-fragment after acid aniline treatment or α-fragment without acid aniline treatment, respectively. This assay is of great importance in the mushroom kingdom, considering the presence of enzymes that are able to hydrolyze phosphodiester bonds (e.g., ribonucleases, ribotoxins and ribotoxin-like proteins) or to remove a specific adenine (rRNA N-glycosylases). Thus, here we used the ß-fragment experimentally detected by Endo's assay as a hallmark to revise the literature available on enzymes from mushrooms and other fungi, whose action consists of protein biosynthesis inhibition.


Assuntos
Agaricales , Ricina , Adenina/metabolismo , Agaricales/metabolismo , Compostos de Anilina , Animais , Proteínas de Plantas/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Ribossômico/análise , RNA Ribossômico/metabolismo , Ratos , Proteínas Inativadoras de Ribossomos/metabolismo , Ribossomos/metabolismo , Ricina/metabolismo
20.
Methods Mol Biol ; 2521: 157-171, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35732997

RESUMO

Due to the lower risks of adverse effects, nonviral gene therapy is a suitable alternative to transfect cancer cells with a suicide gene to let them kill themselves by expressing toxic ribosome-inactivating proteins. Plasmids are stable and easy-to-produce vectors, but they have some disadvantages due to the bacterial backbone. Applying the minicircle technology, this problem can be solved with manageable effort in a well-equipped laboratory. With the described methodology, minicircle-DNA can be produced at low costs. The cell killing properties are monitored following transfection using the CytoSMART® Omni system-a camera based live cell imaging device.


Assuntos
Vetores Genéticos , Proteínas Inativadoras de Ribossomos , Vetores Genéticos/genética , Humanos , Plasmídeos/genética , Ribossomos/genética , Transfecção
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