Prion Strain-Dependent Differences in Conversion of Mutant Prion Proteins in Cell Culture

ABSTRACT Although the protein-only hypothesis proposes that it is the conformation of abnormal prion protein (PrPSc) that determines strain diversity, the molecular basis of strains remains to be elucidated. In the present study, we generated a series of mutations in the normal prion protein (PrPC) in which a single glutamine residue was replaced with a basic amino acid and compared their abilities to convert to PrPSc in cultured neuronal N2a58 cells infected with either the Chandler or 22L mouse-adapted scrapie strain. In mice, these strains generate PrPSc of the same sequence but different conformations, as judged by infrared spectroscopy. Substitutions at codons 97, 167, 171, and 216 generated PrPC that resisted conversion and inhibited the conversion of coexpressed wild-type PrP in both Chandler-infected and 22L-infected cells. Interestingly, substitutions at codons 185 and 218 gave strain-dependent effects. The Q185R and Q185K PrP were efficiently converted to PrPSc in Chandler-infected but not 22L-infected cells. Conversely, Q218R and Q218H PrP were converted only in 22L-infected cells. Moreover, the Q218K PrP exerted a potent inhibitory effect on the conversion of coexpressed wild-type PrP in Chandler-infected cells but had little effect on 22L-infected cells. These results show that two strains with the same PrP sequence but different conformations have differing abilities to convert the same mutated PrPC.

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The inhibitory effect of the ScFv of an anti-prion protein antibody secreted from N2a58 cells on abnormal prion protein accumulation in scrapie-infected cells, ScN2a

Prions ◽

10.1007/4-431-29402-3_54 ◽

2006 ◽

pp. 255-255

Author(s):

Yoshihisa Shimizu ◽

Yuko Kaku-Ushiki ◽

Shigeo Fukuda ◽

Morikazu Shinagawa ◽

Takashi Yokoyama ◽

...

Keyword(s):

Prion Protein ◽

Inhibitory Effect ◽

Protein Accumulation ◽

Infected Cells

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HEPES inhibits the conversion of prion protein in cell culture

Journal of General Virology ◽

10.1099/vir.0.027334-0 ◽

2011 ◽

Vol 92 (5) ◽

pp. 1244-1250 ◽

Cited By ~ 2

Author(s):

Karine Delmouly ◽

Maxime Belondrade ◽

Danielle Casanova ◽

Ollivier Milhavet ◽

Sylvain Lehmann

Keyword(s):

Cell Culture ◽

Prion Protein ◽

Cell Biology ◽

Culture Medium ◽

Inhibitory Effect ◽

Live Cells ◽

Cell Culture Medium ◽

Dependent Inhibition ◽

Infected Cells ◽

Significant Concentration

HEPES is a well-known buffering reagent used in cell-culture medium. Interestingly, this compound is also responsible for significant modifications of biological parameters such as uptake of organic molecules, alteration of oxidative stress mechanisms or inhibition of ion channels. While using cell-culture medium supplemented with HEPES on prion-infected cells, it was noticed that there was a significant concentration-dependent inhibition of accumulation of the abnormal isoform of the prion protein (PrPSc). This effect was present only in live cells and was thought to be related to modification of the PrP environment or biology. These results could modify the interpretation of cell-culture assays of prion therapeutic agents, as well as of previous cell biology results obtained in the field using HEPES buffers. This inhibitory effect of HEPES could also be exploited to prevent contamination or propagation of prions in cell culture.

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Influence of Different Light Regimes on the Mycoparasitic Activity and the Production of the Secondary Metabolite 6-Pentyl-α-Pyrone in Two Strains of Trichoderma Atroviride

10.20944/preprints202009.0251.v1 ◽

2020 ◽

Author(s):

Dubraska Moreno-Ruiz ◽

Alessandro Fuchs ◽

Kristina Missbach ◽

Rainer Schuhmacher ◽

Susanne Zeilinger

Keyword(s):

Secondary Metabolite ◽

Green Light ◽

Inhibitory Effect ◽

Trichoderma Atroviride ◽

Wild Type ◽

Complete Darkness ◽

Light Regimes ◽

Encoding Gene ◽

Type Strains ◽

Strain Dependent

The ascomycete Trichoderma atroviride is well known for its mycoparasitic lifestyle. Similar to other organisms, light is an important cue for T. atroviride. However, besides triggering of conidiation, little is known on the physiological responses of T. atroviride to light. In this study, we analyzed how cultivation under different light wavelengths and regimes impacted the behavior of two T. atroviride wild-type strains, IMI206040 and P1. While colony extension of both strains was slightly affected by light, massive differences in the photoconidation response between the two strains became evident. T. atroviride P1 colonies conidiated under all conditions tested including growth in complete darkness, while IMI206040 required white, blue or green light to trigger asexual reproduction. Interestingly, deletion of the stress-activated MAP kinase-encoding gene tmk3 abolished the ability of strain P1 to conidiate in red and yellow light as well as in darkness. Furthermore, light-dependent differences in the mycoparasitic activity of T. atroviride and in the biosynthesis of the secondary metabolite 6-pentyl--pyrone (6-PP) became evident. 6-PP production was highest upon dark incubation while light, especially exposure to white light as light/dark cycles, had an inhibitory effect on its biosynthesis. We conclude that the response of T. atroviride to light is strain-dependent and impacts differentiation, mycoparasitism and 6-PP production and hence should be considered in experiments testing the mycoparasitic activity of these fungi.

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Orally Administered Amyloidophilic Compound Is Effective in Prolonging the Incubation Periods of Animals Cerebrally Infected with Prion Diseases in a Prion Strain-Dependent Manner

Journal of Virology ◽

10.1128/jvi.01563-07 ◽

2007 ◽

Vol 81 (23) ◽

pp. 12889-12898 ◽

Cited By ~ 71

Author(s):

Yuri Kawasaki ◽

Keiichi Kawagoe ◽

Chun-jen Chen ◽

Kenta Teruya ◽

Yuji Sakasegawa ◽

...

Keyword(s):

Prion Protein ◽

Therapeutic Efficacy ◽

Prion Diseases ◽

Neuroblastoma Cells ◽

Therapeutic Interventions ◽

Dependent Manner ◽

Cell Culture Study ◽

Prion Strain ◽

Incubation Periods ◽

Strain Dependent

ABSTRACT The establishment of effective therapeutic interventions for prion diseases is necessary. We report on a newly developed amyloidophilic compound that displays therapeutic efficacy when administered orally. This compound inhibited abnormal prion protein formation in prion-infected neuroblastoma cells in a prion strain-dependent manner: effectively for RML prion and marginally for 22L prion and Fukuoka-1 prion. When the highest dose (0.2% [wt/wt] in feed) was given orally to cerebrally RML prion-inoculated mice from inoculation until the terminal stage of disease, it extended the incubation periods by 2.3 times compared to the control. The compound exerted therapeutic efficacy in a prion strain-dependent manner such as that observed in the cell culture study: most effective for RML prion, less effective for 22L prion or Fukuoka-1 prion, and marginally effective for 263K prion. Its effectiveness depended on an earlier start of administration. The glycoform pattern of the abnormal prion protein in the treated mice was modified and showed predominance of the diglycosylated form, which resembled that of 263K prion, suggesting that diglycosylated forms of abnormal prion protein might be least sensitive or resistant to the compound. The mechanism of the prion strain-dependent effectiveness needs to be elucidated and managed. Nevertheless, the identification of an orally available amyloidophilic chemical encourages the pursuit of chemotherapy for prion diseases.

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Influence of Different Light Regimes on the Mycoparasitic Activity and 6-Pentyl-α-pyrone Biosynthesis in Two Strains of Trichoderma atroviride

Pathogens ◽

10.3390/pathogens9100860 ◽

2020 ◽

Vol 9 (10) ◽

pp. 860

Author(s):

Dubraska Moreno-Ruiz ◽

Alessandro Fuchs ◽

Kristina Missbach ◽

Rainer Schuhmacher ◽

Susanne Zeilinger

Keyword(s):

Green Light ◽

Inhibitory Effect ◽

Trichoderma Atroviride ◽

Wild Type ◽

Complete Darkness ◽

Yellow Light ◽

Light Regimes ◽

Encoding Gene ◽

Type Strains ◽

Strain Dependent

The ascomycete Trichoderma atroviride is well known for its mycoparasitic lifestyle. Similar to other organisms, light is an important cue for T. atroviride. However, besides triggering of conidiation, little is known on the physiological responses of T. atroviride to light. In this study, we analyzed how cultivation under different light wavelengths and regimes impacted the behavior of two T. atroviride wild-type strains: IMI206040 and P1. While colony extension of both strains was slightly affected by light, massive differences in their photoconidation responses became evident. T. atroviride P1 colonies conidiated under all conditions tested including growth in complete darkness, while IMI206040 required white, blue or green light to trigger asexual reproduction. Interestingly, deletion of the stress-activated MAP kinase-encoding gene tmk3 abolished the ability of strain P1 to conidiate in red and yellow light as well as in darkness. Furthermore, light-dependent differences in the mycoparasitic activity and in the biosynthesis of the secondary metabolite 6-pentyl-α-pyrone (6-PP) became evident. 6-PP production was highest upon dark incubation, while light, especially exposure to white light as light/dark cycles, had an inhibitory effect on its biosynthesis. We conclude that the response of T. atroviride to light is strain-dependent and impacts differentiation, mycoparasitism, and 6-PP production; hence, this should be considered in experiments testing the mycoparasitic activity of these fungi.

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Epitope scanning indicates structural differences in brain-derived monomeric and aggregated mutant prion proteins related to genetic prion diseases

Biochemical Journal ◽

10.1042/bj20130563 ◽

2013 ◽

Vol 454 (3) ◽

pp. 417-425 ◽

Cited By ~ 9

Author(s):

Laura Tapella ◽

Matteo Stravalaci ◽

Antonio Bastone ◽

Emiliano Biasini ◽

Marco Gobbi ◽

...

Keyword(s):

Prion Protein ◽

Prion Proteins ◽

Prion Diseases ◽

Prnp Gene ◽

Structural Heterogeneity ◽

Cellular Prion Protein ◽

Amyloid Angiopathy ◽

Wild Type ◽

Gene Encoding ◽

Jakob Disease

Genetic Creutzfeldt–Jakob disease, Gerstmann–Sträussler–Scheinker syndrome, fatal familial insomnia and prion protein cerebral amyloid angiopathy are clinically and neuropathologically distinct neurodegenerative diseases linked to mutations in the PRNP gene encoding the cellular prion protein (PrPC). How sequence variants of PRNP encode the information to specify these disease phenotypes is not known. It is suggested that each mutation produces a misfolded variant of PrPC with specific neurotoxic properties. However, structural studies of recombinant PrP did not detect major differences between wild-type and mutant molecules, pointing to the importance of investigating mutant PrPs from mammalian brains. We used surface plasmon resonance and a slot-blot immunoassay to analyse the antibody-binding profiles of soluble and insoluble PrP molecules extracted from the brains of transgenic mice modelling different prion diseases. By measuring the reactivity of monoclonal antibodies against different PrP epitopes, we obtained evidence of conformational differences between wild-type and mutant PrPs, and among different mutants. We detected structural heterogeneity in both monomeric and aggregated PrP, supporting the hypothesis that the phenotype of genetic prion diseases is encoded by mutant PrP conformation and assembly state.

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Prion Protein Glycosylation Is Not Required for Strain-Specific Neurotropism

Journal of Virology ◽

10.1128/jvi.02502-08 ◽

2009 ◽

Vol 83 (11) ◽

pp. 5321-5328 ◽

Cited By ~ 41

Author(s):

Justin R. Piro ◽

Brent T. Harris ◽

Koren Nishina ◽

Claudio Soto ◽

Rodrigo Morales ◽

...

Keyword(s):

Prion Protein ◽

Normal Mouse ◽

Cellular Prion Protein ◽

Protein Glycosylation ◽

Wild Type ◽

Prion Strains ◽

Pngase F ◽

Strain Dependent ◽

Normal Mouse Brain ◽

Neuronal Vacuolation

ABSTRACT In this study, we tested the hypothesis that the glycosylation of the pathogenic isoform of the prion protein (PrPSc) might encode the selective neurotropism of prion strains. We prepared unglycosylated cellular prion protein (PrPC) substrate molecules from normal mouse brain by treatment with PNGase F and used reconstituted serial protein cyclic misfolding amplification reactions to produce RML and 301C mouse prions containing unglycosylated PrPSc molecules. Both RML- and 301C-derived prions containing unglycosylated PrPSc molecules were infectious to wild-type mice, and neuropathological analysis showed that mice inoculated with these samples maintained strain-specific patterns of PrPSc deposition and neuronal vacuolation. These results show that PrPSc glycosylation is not necessary for strain-dependent prion neurotropism.

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The GNAQ T96S Mutation Affects Cell Signaling and Enhances the Oncogenic Properties of Hepatocellular Carcinoma

International Journal of Molecular Sciences ◽

10.3390/ijms22063284 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3284

Author(s):

Eugene Choi ◽

Sung Jean Park ◽

Gunhee Lee ◽

Seung Kew Yoon ◽

Minho Lee ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Expression Vector ◽

Signal Transduction Pathway ◽

Inhibitory Effect ◽

The Cancer Genome Atlas ◽

Treatment Modalities ◽

Protein Signaling ◽

Wild Type ◽

Anchorage Independent Growth ◽

Mapk Pathways

Hepatocellular carcinoma (HCC), the most common malignant tumor in the liver, grows and metastasizes rapidly. Despite advances in treatment modalities, the five-year survival rate of HCC remains less than 30%. We sought genetic mutations that may affect the oncogenic properties of HCC, using The Cancer Genome Atlas (TCGA) data analysis. We found that the GNAQ T96S mutation (threonine 96 to serine alteration of the Gαq protein) was present in 12 out of 373 HCC patients (3.2%). To examine the effect of the GNAQ T96S mutation on HCC, we transfected the SK-Hep-1 cell line with the wild-type or the mutant GNAQ T96S expression vector. Transfection with the wild-type GNAQ expression vector enhanced anchorage-independent growth, migration, and the MAPK pathways in the SK-Hep-1 cells compared to control vector transfection. Moreover, cell proliferation, anchorage-independent growth, migration, and the MAPK pathways were further enhanced in the SK-Hep-1 cells transfected with the GNAQ T96S expression vector compared to the wild-type GNAQ-transfected cells. In silico structural analysis shows that the substitution of the GNAQ amino acid threonine 96 with a serine may destabilize the interaction between the regulator of G protein signaling (RGS) protein and GNAQ. This may reduce the inhibitory effect of RGS on GNAQ signaling, enhancing the GNAQ signaling pathway. Single nucleotide polymorphism (SNP) genotyping analysis for Korean HCC patients shows that the GNAQ T96S mutation was found in only one of the 456 patients (0.22%). Our data suggest that the GNAQ T96S hotspot mutation may play an oncogenic role in HCC by potentiating the GNAQ signal transduction pathway.

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Aquatic Toxicity of Photocatalyst Nanoparticles to Green Microalgae Chlorella vulgaris

Water ◽

10.3390/w13010077 ◽

2020 ◽

Vol 13 (1) ◽

pp. 77

Author(s):

Cristina Adochite ◽

Luminita Andronic

Keyword(s):

Chlorella Vulgaris ◽

Advanced Oxidation Process ◽

Growth Parameters ◽

Aquatic Toxicity ◽

Basal Medium ◽

Inhibitory Effect ◽

Synthesis Methods ◽

Potent Inhibitory Effect ◽

Density Surface ◽

The Impact

In the last years, nanoparticles such as TiO2, ZnO, NiO, CuO and Fe2O3 were mainly used in wastewater applications. In addition to the positive aspects concerning using nanoparticles in the advanced oxidation process of wastewater containing pollutants, the impact of these nanoparticles on the environment must also be investigated. The toxicity of nanoparticles is generally investigated by the nanomaterials’ effect on green algae, especially on Chlorella vulgaris. In this review, several aspects are reviewed: the Chlorella vulgaris culture monitoring and growth parameters, the effect of different nanoparticles on Chlorella vulgaris, the toxicity of photocatalyst nanoparticles, and the mechanism of photocatalyst during oxidative stress on the photosynthetic mechanism of Chlorella vulgaris. The Bold basal medium (BBM) is generally recognized as an excellent standard cultivation medium for Chlorella vulgaris in the known environmental conditions such as temperature in the range 20–30 °C and light intensity of around 150 μE·m2·s−1 under a 16/8 h light/dark cycle. The nanoparticles synthesis methods influence the particle size, morphology, density, surface area to generate growth inhibition and further algal deaths at the nanoparticle-dependent concentration. Moreover, the results revealed that nanoparticles caused a more potent inhibitory effect on microalgal growth and severely disrupted algal cells’ membranes.

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An MDM2 inhibitor achieves synergistic cytotoxic effects with adenoviruses lacking E1B55kDa gene on mesothelioma with the wild-type p53 through augmenting NFI expression

Cell Death and Disease ◽

10.1038/s41419-021-03934-y ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Thao Thi Thanh Nguyen ◽

Masato Shingyoji ◽

Michiko Hanazono ◽

Boya Zhong ◽

Takao Morinaga ◽

...

Keyword(s):

Wild Type ◽

Inhibitory Effects ◽

Cytotoxic Effects ◽

P53 Expression ◽

Nuclear Factor I ◽

Infected Cells ◽

Wild Type P53 ◽

Mdm2 Inhibitor ◽

P16 Expression

AbstractA majority of mesothelioma specimens were defective of p14 and p16 expression due to deletion of the INK4A/ARF region, and the p53 pathway was consequently inactivated by elevated MDM2 functions which facilitated p53 degradaton. We investigated a role of p53 elevation by MDM2 inhibitors, nutlin-3a and RG7112, in cytotoxicity of replication-competent adenoviruses (Ad) lacking the p53-binding E1B55kDa gene (Ad-delE1B). We found that a growth inhibition by p53-activating Ad-delE1B was irrelevant to p53 expression in the infected cells, but combination of Ad-delE1B and the MDM2 inhibitor produced synergistic inhibitory effects on mesothelioma with the wild-type but not mutated p53 genotype. The combination augmented p53 phosphorylation, activated apoptotic but not autophagic pathway, and enhanced DNA damage signals through ATM-Chk2 phosphorylation. The MDM2 inhibitors facilitated production of the Ad progenies through augmented expression of nuclear factor I (NFI), one of the transcriptional factors involved in Ad replications. Knocking down of p53 with siRNA did not increase the progeny production or the NFI expression. We also demonstrated anti-tumor effects by the combination of Ad-delE1B and the MDM2 inhibitors in an orthotopic animal model. These data collectively indicated that upregulation of wild-type p53 expression contributed to cytotoxicity by E1B55kDa-defective replicative Ad through NFI induction and suggested that replication-competent Ad together with augmented p53 levels was a therapeutic strategy for p53 wild-type mesothelioma.

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