Characterization, Antioxidant Capacity and Protective Effect of Peptides from Cordyceps militaris Cultivated with Tussah Pupa on Oxidative Injured HepG2 Cells.

The antioxidant capacity and protective effect of peptides from protein hydrolysate of Cordyceps militaris cultivated with tussah pupa (ECPs) on H2O2-injured HepG2 cells were studied. Results indicated ECP1 (<3 kDa) presented the strongest antioxidant activity compared with other molecular weight peptides. Pretreated with ECPs observably enhanced survival rates and reduced apoptosis rates of HepG2 cells. ECPs treatment decreased the ROS level, MDA content and increased CAT and GSH-Px activities of HepG2 cells. Besides, the morphologies of natural peptides from C. militaris cultivated with tussah pupa (NCP1) and ECP1 were observed by scanning electron microscopy (SEM). Characterization results suggested the structure of NCP1 was changed by enzymatic hydrolysis treatment. Most of hydrophobic and acidic amino acids contents (ACC) in ECP1 were also observably improved by enzymatic hydrolysis. In conclusion, low molecular weight peptides had potential value in the development of cosmetics and health food.

Edodin: A New Type of Toxin from Shiitake Mushroom (Lentinula edodes) That Inactivates Mammalian Ribosomes.

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.

Hyphae-specific genes: Possible molecular targets for magnetic iron oxide nanoparticles alone and combined with visible light in Candida albicans.

Candida albicans readily develops resistance to fluconazole. Magnetic iron oxide nanoparticles (denoted as MION) and antimicrobial photodynamic therapy are attracting attention as therapeutic agents. This study aims to investigate the inhibitory efficacy of MION alone and combined with visible light against C. albicans and expression analysis of hyphal wall protein 1 (HWP1) and agglutinin-like sequence 1 (ALS1) genes in C. albicans. Antifungal susceptibility testing, photodynamic activity assay, reactive oxygen species (ROS) production assay and gene expression analysis were determined in C. albicans treated with MION alone and combined with visible light. MION at 1 × minimum inhibitory concentration (MIC) level (500 µg/mL) exhibited antifungal activity against C. albicans isolates. Further, 1 × MIC levels of MION alone and combined with visible light displayed remarkable fungicidal effects at 24 and 48 h after treatment. The MION combined with visible light caused the highest levels of ROS production by all C. albicans isolates. The relative RT-PCR data showed significant downregulation of HWP1 and ALS1 genes which are the key virulence genes in C. albicans. Differences in gene expression of  HWP1 and ALS1 were more significant in MION combined with visible light treatments than MION alone. Our study sheds a novel light on facile development of effective treatment of C. albicans especially fluconazole-resistant C. albicans infections. The hyphae-specific genes HWP1 and ALS1 could be probable molecular targets for MION alone and combined with visible light in C. albicans.

Molecular mechanisms of azole resistance in Candida glabrata isolated from oropharyngeal candidiasis in head and neck cancer patients.

OBJECTIVE: The aim of the current work was to assess the molecular mechanisms of fluconazole-resistant Candida glabrata strains isolated from oropharyngeal candidiasis (OPC) in head and neck patients, as well as evaluation of virulence factors. DESIGN: Antifungal susceptibility pattern of sixty six clinical isolates of C. glabrata were evaluated by broth-microdilution method. The expression of ERG11, CDR1, CDR2, PDR1 genes as well as ERG11 gene capable of possible mutations was also detected in 21 fluconazol-resistant C. glabrata isolates. Phospholipase and proteinase activity of these isolates was estimated, too. The correlation between the virulence factors, antifungal susceptibility patterns and cancer type was also analyzed. RESULTS: Seven synonymous and four non-synonymous mutations were found in 21 fluconazole-resistant C. glabrata isolates; subsequently, four amino acid substitutions including H257P, Q47H, S487Y and I285N were then reported for the first time. High expression of CDR1 and PDR1 in related to other gene findings were tested in these isolates. Additionally, there was no significant difference between stage of cancer and MIC of all antimicrobial drugs. Significant differences between MIC of fluconazole, voriconazole and cancer types were also, found. The proteinase activity (92.4%) was higher than phospholipase activity in the isolates. Further, no significant difference between proteinase (rs: 0.003), phospholipase (rs: -0.107) activity and fluconazole MICs was observed. CONCLUSION: C. glabrata isolated from OPC in head and neck patients represented high capacities for proteolytic enzymes activity and high mRNA level of CDR1 and PDR1 gene and ERG11 mutations play an important role in azole drug resistance.

Fungal antitumor protein D1 is internalized via endocytosis and inhibits non-small cell lung cancer proliferation through MAPK signaling pathway.

Lung cancer has the highest mortality among cancer-related deaths worldwide. Among lung cancers, non-small cell lung cancer (NSCLC) is the most common histological type. In the previous research, we isolated a protein (D1) from Boletus bicolor that inhibits the proliferation of NSCLC cell lines. In this study, we elucidated the internalization mechanism and antitumor mechanism of protein D1 in A549 cells. Protein D1 has a strong inhibitory effect on A549 cells. It binds to secretory carrier membrane protein 3 on the A549 cell membrane and enters A549 cells by clathrin-mediated endocytosis. In vitro, protein D1 activates mitogen-activated protein kinase (MAPK) signaling pathway. JNK and p38MAPK are the biological targets for protein D1. In vivo, protein D1 inhibits the tumor growth of NSCLC xenografts by inducing apoptosis and inhibiting cell proliferation. Protein D1 alters the expression of genes related to apoptosis, cell cycle, and MAPK signaling pathway in tumor cells.

Immunomodulatory protein from ganoderma microsporum protects against oxidative damages and cognitive impairments after traumatic brain injury.

A traumatic brain injury (TBI) causes abnormal proliferation of neuroglial cells, and over-release of glutamate induces oxidative stress and inflammation and leads to neuronal death, memory deficits, and even death if the condition is severe. There is currently no effective treatment for TBI. Recent interests have focused on the benefits of supplements or natural products like Ganoderma. Studies have indicated that immunomodulatory protein from Ganoderma microsporum (GMI) inhibits oxidative stress in lung cancer cells A549 and induces cancer cell death by causing intracellular autophagy. However, no evidence has shown the application of GMI on TBI. Thus, this study addressed whether GMI could be used to prevent or treat TBI through its anti-inflammation and antioxidative effects. We used glutamate-induced excitotoxicity as in vitro model and penetrating brain injury as in vivo model of TBI. We found that GMI inhibits the generation of intracellular reactive oxygen species and reduces neuronal death in cortical neurons against glutamate excitotoxicity. In neurite injury assay, GMI promotes neurite regeneration, the length of the regenerated neurite was even longer than that of the control group. The animal data show that GMI alleviates TBI-induced spatial memory deficits, expedites the restoration of the injured areas, induces the secretion of brain-derived neurotrophic factors, increases the superoxide dismutase 1 (SOD-1) and lowers the astroglial proliferation. It is the first paper to apply GMI to brain-injured diseases and confirms that GMI reduces oxidative stress caused by TBI and improves neurocognitive function. Moreover, the effects show that prevention is better than treatment. Thus, this study provides a potential treatment in naturopathy against TBI.

The polysaccharide-peptide complex from mushroom Cordyceps militaris ameliorates atherosclerosis by modulating the lncRNA-miRNA-mRNA axis.

An N-glycosidic polysaccharide-peptide complex CMPS-80 was obtained from the fruiting body of C. militaris. Of importance, CMPS-80 significantly ameliorated formation of atherosclerotic lesions and plasma lipid profiles in apolipoprotein E-deficient mice. Integrated informatics analysis suggested that CMPS-80 can modulate multiple lncRNA-microRNA-mRNA axes. CMPS-80 has a potential application for prevention of hyperlipidemia and atherosclerosis.

Purification and anticancer activity of glutaminase and urease free intracellular l-asparaginase from Chaetomium sp.

l-asparaginase is a chemotherapeutic drug used in the treatment of acute lymphoblastic leukemia, a malignant disorder in children. l-asparaginase helps in removing acrylamide found in fried and baked foods which is carcinogenic in nature. The search for new therapeutic enzymes is of great interest in both medical and food applications. The present work aims to isolate the intracellular l-asparaginase from endophytic fungi Chaetomium sp. The intracellular enzyme was partially purified by chromatographic techniques. Molecular weight of enzyme was found to be ~66 kDa by SDS PAGE analysis. The enzyme is highly specific for l-asparagine and did not show glutaminase and urease activity. Maximum enzyme activity was found to be 58 ± 5 U/mL at 40 °C, pH 7.0 with 2 µg of protein. Intracellular l-asparaginase from Chaetomium sp. exhibited anticancer activity on human blood cancer (MOLT-4) cells.

Proteins and polysaccharides from vegetative mycelium of medicinal basidiomycete Lentinus edodes display cytotoxicity towards human and animal cancer cell lines.

Detection and study of biologically active compounds seems a promising area of research in cancer diagnostics and therapies. The glycoprotein and polysaccharide fractions showing high cytotoxicity towards several human and animal cancer cell lines: A549, Hep-2, HeLa, С6 and SPEV-2 were isolated from basidiomycete Lentinus edodes vegetative mycelium and fruiting body and further characterized. It was found that water-soluble glycoprotein fractions caused the most significant, 70-100% inhibition of metabolic activity of SPЕV-2, А549 and С6 cell lines. The effective concentrations of glycoprotein fractions reducing the viability of cancer cell lines were determined. The protein and subunit composition of fractions was studied; the highly active galactose-specific lectins were found to be present in these fractions. Comparative analysis of transcriptomes of L. edodes vegetative mycelium, fruiting body and primordium revealed the presence of carbohydrate-binding glycoproteins (lectins) specific for each stage of basidiomycete morphogenesis. Histological examination revealed some morphological indicators of immune system activation and the absence of toxic effect on gastro-intestinal mucosa of animals at peroral administration of fungal glycoprotein fractions. Fungal protein and, in particular, lectin preparations derived from L. еdodes vegetative mycelium might be considered as novel prospective tools in cancer diagnostics and therapies.

Microbial arginine deiminase: A multifaceted green catalyst in biomedical sciences.

Arginine deiminase is a well-recognized guanidino-modifying hydrolase that catalyzes the conversion of L-arginine to citrulline and ammonia. Their biopotential to regress tumors via amino acid deprivation therapy (AADT) has been well established. PEGylated formulation of recombinant Mycoplasma ADI is in the last-phase clinical trials against various arginine-auxotrophic cancers like hepatocellular carcinoma, melanoma, and mesothelioma. Recently, ADIs have attained immense importance in several other biomedical applications, namely treatment of Alzheimer's, as an antiviral drug, bioproduction of nutraceutical L-citrulline and bio-analytics involving L-arginine detection. Considering the wide applications of this biodrug, the demand for ADI is expected to escalate several-fold in the coming years. However, the sustainable production aspects of the enzyme with improved pharmacokinetics is still limited, creating bottlenecks for effective biopharmaceutical development. To circumvent the lacunae in enzyme production with appropriate paradigms of 'quality-by-design' an explicit overview of its properties with 'biobetter' formulations strategies are required. Present review provides an insight into all the potential biomedical applications of ADI along with the improvements required for its reach to clinics. Recent research advances with special emphasis on the development of ADI as a 'biobetter' enzyme have also been comprehensively elaborated.

Cordyceps militaris Immunomodulatory Protein Promotes the Phagocytic Ability of Macrophages through the TLR4-NF-κB Pathway.

Enhancing the phagocytosis of immune cells with medicines provides benefits to the physiological balance by removing foreign pathogens and apoptotic cells. The fungal immunomodulatory protein (FIP) possessing various immunopotentiation functions may be a good candidate for such drugs. However, the effect and mechanism of FIP on the phagocytic activity is limitedly investigated. Therefore, the present study determined effects of Cordyceps militaris immunomodulatory protein (CMIMP), a novel FIP reported to induce cytokines secretion, on the phagocytosis using three different types of models, including microsphere, Escherichia Coli and Candida albicans. CMIMP not only significantly improved the phagocytic ability (p < 0.05), but also enhanced the bactericidal activity (p < 0.05). Meanwhile, the cell size, especially the cytoplasm size, was markedly increased by CMIMP (p < 0.01), accompanied by an increase in the F-actin expression (p < 0.001). Further experiments displayed that CMIMP-induced phagocytosis, cell size and F-actin expression were alleviated by the specific inhibitor of TLR4 (p < 0.05). Similar results were observed in the treatment with the inhibitor of the NF-κB pathway (p < 0.05). In conclusion, it could be speculated that CMIMP promoted the phagocytic ability of macrophages through increasing F-actin expression and cell size in a TLR4-NF-κB pathway dependent way.

Proteomic Research on the Antitumor Properties of Medicinal Mushrooms.

Medicinal mushrooms are increasingly being recognized as an important therapeutic modality in complementary oncology. Until now, more than 800 mushroom species have been known to possess significant pharmacological properties, of which antitumor and immunomodulatory properties have been the most researched. Besides a number of medicinal mushroom preparations being used as dietary supplements and nutraceuticals, several isolates from mushrooms have been used as official antitumor drugs in clinical settings for several decades. Various proteomic approaches allow for the identification of a large number of differentially regulated proteins serendipitously, thereby providing an important platform for a discovery of new potential therapeutic targets and approaches as well as biomarkers of malignant disease. This review is focused on the current state of proteomic research into antitumor mechanisms of some of the most researched medicinal mushroom species, including Phellinus linteus, Ganoderma lucidum, Auricularia auricula, Agrocybe aegerita, Grifola frondosa, and Lentinus edodes, as whole body extracts or various isolates, as well as of complex extract mixtures.

Musarin, a novel protein with tyrosine kinase inhibitory activity from Trametes versicolor, inhibits colorectal cancer stem cell growth.

Colorectal cancer is the second deadly cancer in the world. Trametes versicolor is a traditional Chinese medicinal mushroom with a long history of being used to regulate immunity and prevent cancer. Trametes versicolor mushroom extract demonstrates strongly cell growth inhibitory activity on human colorectal tumor cells. In this study, we characterized a novel 12-kDa protein that named musarin, which was purified from Trametes versicolor mushroom extract and showed significant growth inhibition on multiple human colorectal cancer cell lines in vitro. The protein sequence of musarin was determined through enzyme digestion and MS/MS analysis. Furthermore, Musarin, in particular, strongly inhibits aggressive human colorectal cancer stem cell-like CD24+CD44+ HT29 proliferation in vitro and in a NOD/SCID murine xenograft model. Through whole transcription profile and gene enrichment analysis of musarin-treated CSCs-like cells, major signaling pathways and network modulated by musarin have been enriched, including the bioprocess of the Epithelial-Mesenchymal Transition, the EGFR-Ras signaling pathway and enzyme inhibitor activity. Musarin demonstrated tyrosine kinase inhibitory activity in vitro. Musarin strongly attenuated EGFR expression and down-regulated phosphorylation level, thereby slowing cancer cells proliferation. In addition, oral ingestion of musarin significantly inhibited CD24+CD44+ HT29 generated tumor development in SCID/NOD mice with less side effects in microgram doses. Targeting self-renewal aggressive stem-cell like cancer cell proliferation, with higher water solubility and lower cytotoxicity, musarin has shown strong potence to be developed as a promising novel therapeutic drug candidate against colorectal cancers, especially those that acquire chemo-resistance.

Interrelationship among paraptosis, apoptosis and autophagy in lung cancer A549 cells induced by BEAP, an antitumor protein isolated from the edible porcini mushroom Boletus edulis.

In today's world, cancer is still the leading cause of human death. Among them, the incidence and mortality of lung cancer remain high, and have become the focus of research in the world. BEAP, a protein with anti-lung cancer activity, was isolated and purified from the edible mushroom Boletus edulis. Previous studies have shown that BEAP can inhibit the proliferation of non-small cell lung cancer A549 cells by inducing apoptosis and cell cycle arrest in vitro and in vivo. However, there are many ways in which antitumor proteins from edible and medicinal mushroom play their roles. It is worth exploring whether there are other antitumor mechanisms of BEAP, which can provide reference value for the development of new drugs targeting non-small cell lung cancer and the repurposing of existing drugs.

Microbial production, molecular modification, and practical application of l-Asparaginase: A review.

L-Asparaginase (L-ASNase, EC 3.5.1.1), an antitumor drug for acute lymphoblastic leukemia (ALL) therapy, is widely used in the clinical field. Similarly, L-ASNase is also a powerful and significant biological tool in the food industry to inhibit acrylamide (AA) formation. This review comprehensively summarizes the latest achievements and improvements in the production, modification, and application of microbial L-ASNase. To date, the expression levels and optimization of expression hosts such as Escherichia coli, Bacillus subtilis, and Pichia pastoris, have made significant progress. In addition, examples of successful modification of L-ASNase such as decreasing glutaminase activity, increasing the in vivo stability, and enhancing thermostability have been presented. Impressively, the application of L-ASNase as a food addition aid, as well as its commercialization in the pharmaceutical field, and cutting-edge biosensor application developments have been summarized. The presented results and proposed ideas could be a good guide for other L-ASNase researchers in both scientific and practical fields.

Purification of a novel protein with cytotoxicity against non-small-cell lung cancer cells from Boletus bicolor.

A novel protein (D1 component) was purified from Boletus bicolor by ion-exchange chromatography and gel chromatography on a HiTrap™ Q HP column, a diethylaminoethanol cellulose-52 column, and a Sephadex G75 column, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the D1 component was a single protein band with a molecular weight of about 40 kDa. The sulforhodamine B assay showed that at concentrations as low as 25-75 µg/ml, protein D1 significantly inhibited the proliferation of human lung adenocarcinoma cell lines (A549 and H1299 cells) and had little effect on human normal kidney cells (HEK293 cells). After labeling protein D1, it was found that D1 could enter the cytoplasm of tumor cells and colocated with lysosomes. Flow cytometry results demonstrated that protein D1 induced apoptosis and G1 phase arrest of the cell cycle in A549 and H1299 cells. The Western blot analysis results showed that the expression of apoptosis and cell cycle-related proteins of cleaved caspase-3, cytochrome c, Bax, P16, and P21 was significantly upregulated, whereas the expression of Bcl-2, CDK4, cyclin D, p-Rb, and E2F was significantly downregulated after treatment with protein D1. Therefore, D1 exhibits potential to be developed into an antitumor agent.

Pithohirolide, an antimicrobial tetradepsipeptide from a fungus Pithomyces chartarum.

Pithohirolide (1), a new depsipeptide, was isolated from an ascomycetous fungus Pithomyces chartarum TAMA 581. The planar structure of 1 was elucidated on the basis of NMR and MS analyses and the absolute configuration was determined by the advanced Marfey's analysis, chiral-phase HPLC analysis, and synthesis of degradation product. Compound 1 possesses a cyclic structure comprising (S)-2-hydroxy-3-phenylpropanoic acid, (S)-3-hydroxy-3-phenylpropanoic acid, (S)-2-hydroxyisovaleric acid, and N-methyl-L-alanine, connected via three ester and one amide linkages. Compound 1 exhibited antimicrobial activity against Staphylococcus aureus and Saccharomyces cerevisiae at MIC 3.1 µg ml-1.

The chaperone-like protein Cdc48 regulates ubiquitin-proteasome system in plants.

The degradation of misfolded proteins is mainly mediated by the ubiquitin-proteasome system (UPS). UPS can be assisted by the protein Cdc48 but the relationship between UPS and Cdc48 in plants has been poorly investigated. Here, we analysed the regulation of UPS by Cdc48 in tobacco thanks to two independent cell lines overexpressing Cdc48 constitutively and plant leaves overexpressing Cdc48 transiently. In the cell lines, the accumulation of ubiquitinated proteins was affected both quantitatively and qualitatively and the number of proteasomal subunits was modified, while proteolytic activities were unchanged. Similarly, the over-expression of Cdc48 in planta impacted the accumulation of ubiquitinated proteins. A similar process occurred in leaves overexpressing transiently Rpn3, a proteasome subunit. Cdc48 being involved in plant immunity, its regulation of UPS was also investigated in response to cryptogein, an elicitor of immune responses. In the cell lines stably overexpressing Cdc48 and in leaves transiently overexpressing Cdc48 and/or Rpn3, cryptogein triggered a premature cell death while no increase of the proteasomal activity occurred. Overall, this study highlights a role for Cdc48 in ubiquitin homeostasis and confirms its involvement, as well as that of Rpn3, in the processes underlying the hypersensitive response.

Potential mechanisms underlying the protective effects of Tricholoma matsutake singer peptides against LPS-induced inflammation in RAW264.7 macrophages.

This study aimed to investigate the protective effects of a < 3 kDa Tricholoma matsutake Singer peptide (TMWP) on lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophages. The results showed that TMWP significantly upregulated superoxide dismutase (SOD) activity and reduced reactive oxygen species (ROS) generation in RAW264.7 macrophages. Western blotting and immunofluorescence analysis indicated that TMWP inhibited the activation of the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways, thereby reducing the secretion of IL-1ß and IL-6 and the expression of TNF-α, COX-2, and iNOS. Additionally, TMWP improved mitochondrial respiration in LPS-stressed macrophages, counteracting the harmful effects of LPS treatment on mitochondrial function. Three peptides (SDIKHFPF, SDLKHFPF, and WFNNAGP) with the highest predicted scores for potential anti-inflammatory activity were identified using nano-HPLC-MS/MS. These data indicated that T. matsutake peptides could be an attractive natural ingredient for developing novel functional foods.

Fungal natural products galaxy: Biochemistry and molecular genetics toward blockbuster drugs discovery.

Secondary metabolites synthesized by fungi have become a precious source of inspiration for the design of novel drugs. Indeed, fungi are prolific producers of fascinating, diverse, structurally complex, and low-molecular-mass natural products with high therapeutic leads, such as novel antimicrobial compounds, anticancer compounds, immunosuppressive agents, among others. Given that these microorganisms possess the extraordinary capacity to secrete diverse chemical scaffolds, they have been highly exploited by the giant pharma companies to generate small molecules. This has been made possible because the isolation of metabolites from fungal natural sources is feasible and surpasses the organic synthesis of compounds, which otherwise remains a significant bottleneck in the drug discovery process. Here in this comprehensive review, we have discussed recent studies on different fungi (pathogenic, non-pathogenic, commensal, and endophytic/symbiotic) from different habitats (terrestrial and marines), the specialized metabolites they biosynthesize, and the drugs derived from these specialized metabolites. Moreover, we have unveiled the logic behind the biosynthesis of vital chemical scaffolds, such as NRPS, PKS, PKS-NRPS hybrid, RiPPS, terpenoids, indole alkaloids, and their genetic mechanisms. Besides, we have provided a glimpse of the concept behind mycotoxins, virulence factor, and host immune response based on fungal infections.