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BACKGROUND: The mortality rate of liver cancer ranks third in the world, and hepatocellular carcinoma (HCC) is a malignant tumor of the digestive tract. Cucurbitacin B (CuB), a natural compound extracted from Cucurbitaceae spp., is the main active component of Chinese patent medicine the Cucurbitacin Tablet, which has been widely used in the treatment of various malignant tumors in clinics, especially HCC. PURPOSE: This study explored the role and mechanism of CuB in the suppression of liver cancer progression. METHODS: Cell Counting Kit-8 (CCK-8) and colony formation assays were used to detect the inhibitory function of CuB in Huh7, Hep3B, and Hepa1/6 hepatoma cells. Calcein-AM/propidium iodide (PI) staining and lactate dehydrogenase (LDH) measurement assays were performed to determine cell death. Mitochondrial membrane potential (Δψm) was measured, and flow cytometry was performed to evaluate cell apoptosis and cell cycle. Several techniques, such as proteomics, Western blotting (WB), and ribonucleic acid (RNA) interference, were utilized to explore the potential mechanism. The animal experiment was performed to verify the results of in vitro experiments. RESULTS: CuB significantly inhibited the growth of Huh7, Hep3B, and Hepa1/6 cells and triggered the cell cycle arrest in G2/M phage without leading to cell death, especially apoptosis. Knockdown of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), a target of CuB, did not reverse CuB elicited cell cycle arrest. CuB enhanced phosphorylated ataxia telangiectasia mutated (p-ATM) and phosphorylated H2A histone family member X (γ-H2AX) levels. Moreover, CuB increased p53 and p21 levels and decreased cyclin-dependent kinase 1 (CDK1) expression, accompanied by improving phosphorylated checkpoint kinase 1 (p-CHK1) level and suppressing cell division cycle 25C (CDC25C) protein level. Interestingly, these phenomena were partly abolished by a deoxyribonucleic acid (DNA) protector methylproamine (MPA). Animal studies showed that CuB also significantly suppressed tumor growth in BALB/c mice bearing Hepa1/6 cells. In tumor tissues, CuB reduced the expression levels of proliferating cell nuclear antigen (PCNA) and γ-H2AX but did not change the terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) level. CONCLUSION: This study demonstrated for the first time that CuB could effectively impede HCC progression by inducing DNA damage-dependent cell cycle arrest without directly triggering cell death, such as necrosis and apoptosis. The effect was achieved through ataxia telangiectasia mutated (ATM)-dependent p53-p21-CDK1 and checkpoint kinase 1 (CHK1)-CDC25C signaling pathways. These findings indicate that CuB may be used as an anti-HCC drug, when the current findings are confirmed by independent studies and after many more clinical phase 1, 2, 3, and 4 testings have been done.
Assuntos
Ataxia Telangiectasia , Carcinoma Hepatocelular , Neoplasias Hepáticas , Triterpenos , Animais , Camundongos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Quinase 1 do Ponto de Checagem/uso terapêutico , Proteína Supressora de Tumor p53/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/uso terapêutico , Pontos de Checagem do Ciclo Celular , Dano ao DNA , Apoptose , Linhagem Celular Tumoral , Proliferação de CélulasRESUMO
BACKGROUND: Huachansu (HCS), a known Chinese patent drug extracted from the Chinese toad skin, is frequently used for the treatment of various advanced cancers, especially gastric cancer, due to the good therapeutic effect. However, it is rather difficult to clarify the active substances and molecular mechanisms involved owing to the lack of appropriate research strategies. We recently proposed the concept and research ideas of compound-composed Chinese medicine formula. PURPOSE: To discover compound-composed Chinese medicine from Huachansu and to explore its mechanism of action in inducing apoptosis of gastric cancer cells. METHOD: Network pharmacology combined with serum pharmacochemistry was utilized to screen the predominant active constituents from HCS against gastric cancer. Then, the compound-composed Chinese medicine of HCS (CCMH) was prepared according to their relative contents in serum. The pharmacological effects and potential mechanisms for CCMH were investigated by assays for cell viability, cell cycle, apoptosis, mitochondrial membrane potential (MMP), proteomics, reactive oxygen species (ROS), N-Acetylcysteine (NAC) antagonism, proteasome activity, and western blot. RESULTS: CCMH was comprised of arenobufagin (11.14%), bufalin (18.67%), bufotalin (7.33%), cinobufagin (16.67%), cinobufotalin (16.74%), gamabufotalin (8.45%), resibufogenin (12.03%), and telocinobufagin (8.97%). CCMH evidently induced proliferation inhibition, cell cycle arrest, apoptosis, and MMP collapse in gastric cancer cells, possessing the better activities than HCS. Proteomic analysis showed that CCMH influenced ROS pathway, ubiquitin proteasome system, and PI3K/Akt and MAPK signaling pathways. CCMH markedly enhanced intracellular ROS levels in gastric cancer cells, which was reversed by NAC. Accordingly, NAC antagonized the apoptosis-inducing effect of CCMH. Significantly decreased proteasome 20S activity by CCMH was observed in gastric cancer cells. CCMH also regulated the expression of key proteins in PI3K/Akt and MAPK signaling pathways. CONCLUSION: CCMH possesses more significant apoptotic induction effects on gastric cancer cells than HCS, which is achieved primarily through suppression of proteasome activities and increase of ROS levels, followed by regulating PI3K/Akt and MAPK signaling pathways. Network pharmacology combined with serum pharmacochemistry is an effective strategy for discovering compound-composed Chinese medicine from traditional Chinese medicine, which can help clarify the pharmacological substances and mechanisms of action for traditional Chinese medicine.
Assuntos
Venenos de Anfíbios , Neoplasias Gástricas , Humanos , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas Proto-Oncogênicas c-akt/metabolismo , Medicina Tradicional Chinesa , Fosfatidilinositol 3-Quinases/metabolismo , Proteômica , Linhagem Celular Tumoral , ApoptoseRESUMO
Proteins do not only serve as nutrients to fulfill the demand for food, but also are used as a source of bioactive proteins/polypeptides for regulating physical functions and promoting physical health. Female breast cancer has the highest incidence in the world and is a serious threat to women's health. Bioactive proteins/polypeptides exert strong anti-tumor effects and exhibit inhibition of multiple breast cancer cells. This review discussed the suppressing effects of bioactive proteins/polypeptides on breast cancer in vitro and in vivo, and their mechanisms of migration and invasion inhibition, apoptosis induction, and cell cycle arrest. This may contribute to providing a basis for the development of bioactive proteins/polypeptides for the treatment of breast cancer.
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Sesamum indicum is a major and important oilseed crop that is believed to promote human health in many countries, especially in China. Sesame seeds contain two types of lignans: lipid-soluble lignans and water-soluble glucosylated lignans. The major glucosylated lignans are sesaminol glucosides (SGs). So far, four sesaminol isomers and four SGs are identified. During the naturally occurring process of SGs production, sesaminol is generated first from two molecules of E-coniferyl alcohol, and then the sugar is added to the sesaminol one by one, leading to production of SGs. Sesaminol can be prepared from SGs, from sesamolin, and through artificial synthesis. SGs are metabolized in the liver and intestine and are then transported to other tissues. They exhibit several biological activities, most of which are based on their antioxidant and anti-inflammatory activities. In this paper, we present an overview of the current status of research on sesaminol and SGs. We have also discussed their synthesis, preparation, metabolism, and biological activities. It has been suggested that sesaminol and SGs are important biological substances with strong antioxidant properties in vitro and in vivo and are widely used in the food industry, medicine, and cosmetic products. The recovery and utilization of SGs from sesame seed cake after oil processing will generate massive economic benefits.
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Spermine, spermidine and putrescine polyamines are naturally occurring ubiquitous positively charged amines and are essential metabolites for biological functions in our life. These compounds play a crucial role in many cell processes, including cellular proliferation, growth, and differentiation. Intracellular levels of polyamines depend on their biosynthesis, transport and degradation. Polyamine levels are high in cancer cells, which leads to the promotion of tumor growth, invasion and metastasis. Targeting polyamine metabolism as an anticancer strategy is considerably rational. Due to compensatory mechanisms, a single strategy does not achieve satisfactory clinical effects when using a single agent. Combination regimens are more clinically promising for cancer chemoprevention because they work synergistically with causing little or no adverse effects due to each individual agent being used at lower doses. Moreover, bioactive substances have advantages over single chemical agents because they can affect multiple targets. In this review, we discuss anticancer strategies targeting polyamine metabolism and describe how combination treatments and effective natural active ingredients are promising therapies. The existing research suggests that polyamine metabolic enzymes are important therapeutic targets and that combination therapies can be more effective than monotherapies based on polyamine depletion.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Homeostase/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Poliaminas/antagonistas & inibidores , Poliaminas/metabolismo , Animais , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Humanos , Poliaminas/químicaRESUMO
The global health emergency generated by coronavirus disease-2019 has prompted the search for immunomodulatory agents. There are many potential natural products for drug discovery and development to tackle this disease. One of these candidates is the Ganoderma lucidum fungal immunomodulatory protein (FIP-glu). In the present study, we clarify the influences of N-linked glycans on the improvement of anti-inflammatory activity and the potential mechanisms of action. Four proteins, including FIP-glu (WT) and its mutants N31S, T36N and N31S/T36N, were successfully expressed in P. pastoris, of which T36N and N31S/T36N were glycoproteins. After treatment with peptide-N-glycosidase F, the results of SDS-PAGE and Western blot showed that the glycan moiety was removed completely, indicating that the glycan moiety was N-linked. This was also demonstrated by UPLC-qTOF-MS. The cytotoxicity assay showed that N-linked glycans decreased the cytotoxicity of WT; while, the RT-qPCR assay showed that N-glycosylated WT regulated the mRNA expression of IL-6 and TGF-ß1. The Western blot results showed that N-glycosylated WT reduced the phosphorylation level of p38 MAPK. In conclusion, our findings revealed a novel mechanism by which N-glycosylation of FIP-glu improved its anti-inflammatory activity through the regulation of the expression of inflammatory cytokines in RAW264.7 via inhibition of p38 MAPK phosphorylation. It was proved that N-glycosylation significantly improved the functional properties of FIP-glu, providing theoretical and technical support for expanding the application of FIPs in the food and pharmaceutical industries.
Assuntos
Proteínas Fúngicas/farmacologia , Fatores Imunológicos/farmacologia , Imunomodulação/efeitos dos fármacos , Reishi , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , Citocinas , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/metabolismo , Glicosilação , Espectrometria de Massas , Camundongos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Células RAW 264.7 , Reação em Cadeia da Polimerase em Tempo Real , SaccharomycetalesRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Chinese Cordyceps (DongChong XiaCao), a parasitic complex of a fungus Ophiocordyceps sinensis and a caterpillar, is a traditional Chinese medicine. Polysaccharides extracted from O. sinensis have immunomodulatory effects on macrophages. However, the mechanism of polysaccharides on macrophage and the composition of polysaccharides are not known. AIM OF STUDY: We aimed to investigate composition and structure of the intracellular polysaccharides from O. sinensis mycelia (designed as OSP), and evaluate its the immunomodulatory effect on macrophages and its underlying mechanism. MATERIALS AND METHODS: We performed a liquid-state fermentation of O. sinensis to produce mycelia. The DEAE-Sephadex-A25 cellulose column and Sephadex-G100 gel column chromatography were employed to purify and character the intracellular OSP. Macrophages RAW264.7 cells were employed to evaluate OSP's immunomodulatory activity and the possible mechanism responsible for the activation of macrophages in vitro. RESULTS: The average molecular weight of OSP was distributed at 27,972 Da, OSP was composed of xylose, mannose, glucose, and galactose with the ratio of 2.9 : 6.6 : 166 : 2.6, with a trace amount of fucose, arabinose and rhamnose. The phagocytosis of RAW264.7 cells was improved significantly and remarkable changes were observed in the morphology with OSP-treated cells. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis demonstrated that OSP had an ability to regulate the mRNA expression of pro-inflammatory and anti-inflammatory cytokines, and to induce the mRNA expression level of iNOS in a concentration dependent manner in RAW264.7 cells. Western blotting analysis showed that the regulation of NO and cytokines was mediated through mitogen-activated protein kinase (MAPK) and PI3K/Akt signaling pathways. CONCLUSION: This study demonstrated that OSP was with a capacity to activate macrophage cells RAW264.7 for an improvement of immunomodulation activities, which was through regulation of inflammatory mediators via MAPK and PI3K/Akt signaling pathways.
Assuntos
Cordyceps/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Polissacarídeos/imunologia , Polissacarídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Espaço Intracelular/química , Lipopolissacarídeos/toxicidade , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Monossacarídeos/análise , Micélio/química , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fagocitose/efeitos dos fármacos , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Células RAW 264.7RESUMO
Ganoderma lucidum immunomodulatory protein (FIP-glu) is an active ingredient with potential immunoregulatory functions. The study was conducted to explore the immunomodulatory activities of recombinant FIP-glu (rFIP-glu) and its possible mechanism in macrophage RAW264.7 cells. In vitro assays of biological activity indicated that rFIP-glu significantly activated RAW264.7 cells and possessed proinflammatory and anti-inflammatory abilities. RNA sequencing analysis and Western blot analysis showed that macrophage activation involved PI3K/Akt and MAPK pathways. Furthermore, real-time quantitative polymerase chain reaction indicated that the PI3K inhibitor LY294002 blocked the messenger RNA (mRNA) levels of MCP-1 (CCL-2), the MEK1/2 inhibitor U0126 reduced the mRNA levels of TNF-α and MCP-1 (CCL-2), and the JNK1/2/3 inhibitor SP600125 prevented the upregulation of inducible nitric oxide synthase mRNA in rFIP-glu-induced cells. rFIP-glu did not mediate these inflammatory effects through a general pathway but rather through a different pathway for a different inflammatory mediator. These data imply that rFIP-glu possessed immunomodulatory activity in macrophages, which was mediated through PI3K/Akt and MAPK pathways.
Assuntos
Proteínas Fúngicas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Reishi , Animais , Imunomodulação/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células RAW 264.7RESUMO
During recent decades, >30 fungal immunomodulatory proteins (FIPs) have been found in a range of mushrooms and other fungi. Various pharmacological functions of FIPs have become important in the discovery and development of new drugs. In this review, we discuss some important factors, focusing on the use of amino acid sequence data to predict structural and physicochemical properties. We also discuss pharmacologic activities and possible mechanisms of the proteins with a focus on antitumor activities. Numerous other questions must also be addressed before FIPs can be widely accepted and used as antitumor agents.
Assuntos
Antineoplásicos , Proteínas Fúngicas , Fatores Imunológicos , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacologia , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/farmacologiaRESUMO
Ganoderma mushrooms for medicinal use contain various bioactive compounds, but the genetic elements available for these medicinal mushrooms are still limited. In this study we cloned and analyzed the promoters of fungal immunomodulatory protein (FIP) genes from G. lucidum and G. atrum. FIP gene expression was induced by different concentrations of methyl jasmonate (MeJA) and salicylic acid (SA), and messenger RNA expression was detected by quantitative reverse-transcription polymerase chain reaction. The results provided 5' upstream sequences of FIP genes from G. lucidum and G. atrum. Sequence analysis showed that the FIP-glu promoter sequence contained 11 CAAT boxes, 3 TATA boxes, 3 MeJA-responsive elements, 3 MYB binding site (MBS) motifs, 1 abscisic acid responsive element, 1 TGA, 1 anaerobic inducible element, 2 circadian elements, 1 fungal elicitor, 1 meristem-specific activation element, 3 Skn-1 motifs, and several light-responsive elements. The 5' flanking region of FIP-gat included 9 CAAT boxes, 4 TATA boxes, 3 MeJA-responsive elements, 1 AuxRR core, 1 GC motif, 1 MBS, 1 fungal elicitor, 1 meristem-specific activation element, 3 Skn-1 motifs, and several light-responsive elements. On the transcriptional level, both FIP-glu and FIP-gat reached their highest expression after treatment with MeJA at 500 µmol/L. FIP-glu expression depended on the concentration of SA (0-1000 mg/L); the expression of the FIP-gat gene was highest at a concentration of 100 mg MeJA/L. This research lays the foundation to use Ganoderma mycelia as bioreactors for producing FIPs.
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Proteínas Fúngicas/genética , Ganoderma/genética , Regulação Fúngica da Expressão Gênica , Fatores Imunológicos/genética , Regiões Promotoras Genéticas/genética , Acetatos/farmacologia , Antifúngicos/farmacologia , Sequência de Bases , Sítios de Ligação , Clonagem Molecular/métodos , Ciclopentanos/farmacologia , Genes Fúngicos , Oxilipinas/farmacologia , Ácido Salicílico/farmacologia , Análise de Sequência de DNARESUMO
Fungal immunomodulatory protein (FIP), extracted from higher basidiomycetes, is a kind of small molecule protein with extensive biological functions, including anti-tumor and anti-allergy, stimulating immune cells to produce a variety of cytokines, etc. Compared with FIP-glu, FIP-SN15, a novel gene shuffled from the genes of Ganoderma sinensis and Ganoderma lucidum FIP, was used as the object in this study. Based on the construction of prokaryotic expression vectors, both pET30a-FIP-glu and pET30a-FIP-SN15 were expressed in Escherichia coli. Then the recombinant proteins are respectively analyzed by Western blot, Q-TOF MS, and bioinformatics techniques. Finally, effects of reFIPs on cell cycle and apoptosis of human glioblastoma cell line U-251 MG were studied by fluorescence activated cell sorting (FACS). The results showed that the recombinant proteins FIP-SN15 and FIP-glu could be successfully expressed in E. coli, the yield of which was 35.95 and 36.67 mg/L, respectively. The recombinant protein FIP-SN15 consisted of 111 amino acids, and four peptides were identified by Q-TOF MS with a coverage of 91.9 %. The secondary and tertiary structure of FIP-SN15 were also predicted by bioinformatics method which suggest that reFIP-SN15 was a new member of FIPs family. FACS analysis showed that 10 µg/mL FIP-SN15 and FIP-glu could induce U-251 MG cells apoptosis, the apoptotic rates were increased by 6.03 and 22.01 %, respectively. The results of reFIPs on U-251 MG cell cycle indicated that reFIPs could inhibit cell cycle progression by retardation of G1/S transition. The efforts in this assay would lay the foundation for further development of reFIPs products and research on the anti-tumor mechanisms of FIP-SN15.
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Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Apoptose , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Clonagem Molecular , Embaralhamento de DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/farmacologia , Ganoderma/genética , Expressão Gênica , Humanos , Neuroglia/efeitos dos fármacos , Neuroglia/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologiaRESUMO
Fugal immunomodulatory protein from Flammulina velutipes (FIP-fve) belongs to FIPs family, which has precious pharmaceutical value. To understand the regulatory mechanism of FIP-fve expression, we have cloned a 900 bp genomic DNA fragment from the transcriptional start site of the FIP-fve gene using genomic walker technology. Sequence analysis showed the presence of several eukaryotic transcription factor binding motifs in the 900 bp of upstream region of the FIP-fve gene, which contains one putative TATA-boxes, four possible CAAT-boxes, one ABRE, one ARE, three CGTCA-motifs, two TGA-elements and four Skn-1 motifs. The eukaryotic expression vector pfveP:: GUS-GFP was transferred into tobacco via an agrobacterium-mediated leaf disc transformation. The results showed that the FIP-fve promoter could induce the reporter gene GUS or GFP expression in different tissues of tobaccos. This study would lay a foundation for expression regulation of FIP-fve and development of genetic-modified plant products.
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Flammulina/genética , Carpóforos/genética , Proteínas Fúngicas/genética , Fatores Imunológicos/genética , Nicotiana/genética , Regiões Promotoras Genéticas , Agrobacterium tumefaciens/genética , Sítios de Ligação , Clonagem Molecular , Flammulina/química , Flammulina/metabolismo , Carpóforos/química , Carpóforos/metabolismo , Proteínas Fúngicas/metabolismo , Técnicas de Transferência de Genes , Genes Reporter , Vetores Genéticos , Fatores Imunológicos/metabolismo , Dados de Sequência Molecular , Motivos de Nucleotídeos , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Nicotiana/metabolismoRESUMO
Fungal immunomodulatory proteins (FIPs) found in a wide variety of mushrooms hold significant therapeutic potential. Despite much research, the structural determinants for their immunomodulatory functions remain unknown. In this study, a DNA shuffling technique was used to create two shuffled FIP protein libraries: an intrageneric group containing products of shuffling between FIP-glu (FIP gene isolated from Ganoderma lucidum) and FIP-gsi (FIP gene isolated from Ganoderma sinense) genes and an intergeneric group containing the products of shuffling between FIP-glu, FIP-fve (FIP gene isolated from Flammulina velutipes), and FIP-vvo (FIP gene isolated from Volvariella volvacea) genes. The gene shuffling generated 426 and 412 recombinant clones, respectively. Using colony blot analysis, we selected clones that expressed relatively high levels of shuffled gene products recognized by specific polyclonal antibodies. We analyzed the DNA sequences of the selected shuffled genes, and testing of their protein products revealed that they maintained functional abilities to agglutinate blood cells and induce cytokine production by splenocytes from Kunming mice in vitro. Meanwhile, the relationships between protein structure and the hemagglutination activity and between the changed nucleotide sites and expression levels were explored by bioinformatic analysis. These combined analyses identified the nucleotide changes involved in regulating the expression levels and hemagglutination activities of the FIPs. Therefore, we were able to generate recombinant FIPs with improved biological activities and expression levels by using DNA shuffling, a powerful tool for the generation of novel therapeutic proteins and for their structural and functional studies.
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Embaralhamento de DNA/métodos , Evolução Molecular Direcionada/métodos , Proteínas Fúngicas/genética , Proteínas Fúngicas/imunologia , Fatores Imunológicos/genética , Fatores Imunológicos/imunologia , Sequência de Aminoácidos , Animais , Citocinas/metabolismo , Flammulina/genética , Flammulina/imunologia , Ganoderma/genética , Ganoderma/imunologia , Hemaglutinação , Leucócitos Mononucleares/imunologia , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Análise de Sequência de DNA , Volvariella/genética , Volvariella/imunologiaRESUMO
A Cu/Zn-superoxide dismutase (SOD) gene was characterized from Cordycepes militaris by gene cloning, heterogeneous expression and function analysis. This 154-aa SOD (CmSOD) was deduced from a 465-bp gene cloned, showing 72-95 % sequence identity to Cu/Zn-SODs from other fungi. The deduced amino acid sequence of the cDNA is highly similar to Beauveria bassiana (95 %), Isaria tenuipes (94 %) and Claviceps purpurea (88 %), respectively. The SOD gene of C. militaris spin 589 bp and consisted of two introns and three exons. The CmSOD coding region sequence was inserted into plasmid pQE-30 in order to construct prokaryotic expression vector, then transformed into Escherichia coli M15 cells for expression, and a mass of rCmSOD was obtained by IPTG induction. The enzyme activity of the purified rCmSOD was approximately 714.48 U/mg after the assay. The study provided a way for in-depth research on the expression and regulation of the CmSOD, and the molecular mechanism of anti-oxidative effect in C. militaris.
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Cordyceps/enzimologia , Proteínas Fúngicas/metabolismo , Superóxido Dismutase/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Escherichia coli , Éxons , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Expressão Gênica , Insetos/microbiologia , Íntrons , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Superóxido Dismutase/química , Superóxido Dismutase/genética , Superóxido Dismutase/isolamento & purificaçãoRESUMO
With the exception of polysaccharides and triterpenes/triterpenoids compounds, fungal immunomodulatory protein (FIP), a small molecule protein, is also an important bioactive component with immune regulating activity. It plays a significant role in immunomodulating. The objective of this paper was to review the latest advances in various aspects of research on FIPs, including their basic components and structural character, characters of diversity, gene cloning and expression, and their biological function, etc. In addition, prospects of utilization value and the exploitation foreground of FIPs were also discussed. The review will provide a useful reference for further research, development, and utilizations of FIPs.