scholarly journals Characterization of Anchorless Human PrP With Q227X Stop Mutation Linked to Gerstmann-Sträussler-Scheinker Syndrome In Vivo and In Vitro

2020 ◽  
Vol 58 (1) ◽  
pp. 21-33
Author(s):  
Pingping Shen ◽  
Johnny Dang ◽  
Zerui Wang ◽  
Weiguanliu Zhang ◽  
Jue Yuan ◽  
...  
Keyword(s):  
Protein Misfolding ◽  
Neuroblastoma Cells ◽  
Cellular Prion Protein ◽  
Wild Type ◽  
Gpi Anchor ◽  
Two Dimensional Gel Electrophoresis ◽  
Molecular Weights ◽  
Prion Propagation

AbstractAlteration in cellular prion protein (PrPC) localization on the cell surface through mediation of the glycosylphosphatidylinositol (GPI) anchor has been reported to dramatically affect the formation and infectivity of its pathological isoform (PrPSc). A patient with Gerstmann-Sträussler-Scheinker (GSS) syndrome was previously found to have a nonsense heterozygous PrP-Q227X mutation resulting in an anchorless PrP. However, the allelic origin of this anchorless PrPSc and cellular trafficking of PrPQ227X remain to be determined. Here, we show that PrPSc in the brain of this GSS patient is mainly composed of the mutant but not wild-type PrP (PrPWt), suggesting pathological PrPQ227X is incapable of recruiting PrPWt in vivo. This mutant anchorless protein, however, is able to recruit PrPWt from humanized transgenic mouse brain but not from autopsied human brain homogenates to produce a protease-resistant PrPSc-like form in vitro by protein misfolding cyclic amplification (PMCA). To further investigate the characteristics of this mutation, constructs expressing human PrPQ227X or PrPWt were transfected into neuroblastoma cells (M17). Fractionation of the M17 cells demonstrated that most PrPWt is recovered in the cell lysate fraction, while most of the mutant PrPQ227X is recovered in the medium fraction, consistent with the results obtained by immunofluorescence microscopy. Two-dimensional gel-electrophoresis and Western blotting showed that cellular PrPQ227X spots clustered at molecular weights of 22–25 kDa with an isoelectric point (pI) of 3.5–5.5, whereas protein spots from the medium are at 18–26 kDa with a pI of 7–10. Our findings suggest that the role of GPI anchor in prion propagation between the anchorless mutant PrP and wild-type PrP relies on the cellular distribution of the protein.

scholarly journals Overexpression of quality control proteins reduces prion conversion in prion-infected cells

2018 ◽  
Vol 293 (41) ◽  
pp. 16069-16082 ◽  
Author(s):  
Simrika Thapa ◽  
Basant Abdulrahman ◽  
Dalia H. Abdelaziz ◽  
Li Lu ◽  
Manel Ben Aissa ◽  
...  
Keyword(s):  
Quality Control ◽  
De Novo ◽  
Prion Diseases ◽  
Neuroblastoma Cells ◽  
Cellular Prion Protein ◽  
Control Mechanisms ◽  
Cellular Mechanisms ◽  
Prion Propagation ◽  
Infected Cells

Prion diseases are fatal infectious neurodegenerative disorders in humans and other animals and are caused by misfolding of the cellular prion protein (PrPC) into the pathological isoform PrPSc. These diseases have the potential to transmit within or between species, including zoonotic transmission to humans. Elucidating the molecular and cellular mechanisms underlying prion propagation and transmission is therefore critical for developing molecular strategies for disease intervention. We have shown previously that impaired quality control mechanisms directly influence prion propagation. In this study, we manipulated cellular quality control pathways in vitro by stably and transiently overexpressing selected quality control folding (ERp57) and cargo (VIP36) proteins and investigated the effects of this overexpression on prion propagation. We found that ERp57 or VIP36 overexpression in persistently prion-infected neuroblastoma cells significantly reduces the amount of PrPSc in immunoblots and prion-seeding activity in the real-time quaking-induced conversion (RT-QuIC) assay. Using different cell lines infected with various prion strains confirmed that this effect is not cell type– or prion strain–specific. Moreover, de novo prion infection revealed that the overexpression significantly reduced newly formed PrPSc in acutely infected cells. ERp57-overexpressing cells significantly overcame endoplasmic reticulum stress, as revealed by expression of lower levels of the stress markers BiP and CHOP, accompanied by a decrease in PrP aggregates. Furthermore, application of ERp57-expressing lentiviruses prolonged the survival of prion-infected mice. Taken together, improved cellular quality control via ERp57 or VIP36 overexpression impairs prion propagation and could be utilized as a potential therapeutic strategy.

scholarly journals Midkine Mediates Intercellular Crosstalk between Drug-Resistant and Drug-Sensitive Neuroblastoma Cells In Vitro and In Vivo

ISRN Oncology ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-12
Author(s):  
Fei Chu ◽  
Jessica A. Naiditch ◽  
Sandra Clark ◽  
Yi-Yong Qiu ◽  
Xin Zheng ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Chemotherapy Resistance ◽  
Neuroblastoma Cells ◽  
Cytotoxic Agents ◽  
Drug Resistant ◽  
Wild Type ◽  
Cytoprotective Effect ◽  
Acquired Drug Resistance

Resistance to cytotoxic agents has long been known to be a major limitation in the treatment of human cancers. Although many mechanisms of drug resistance have been identified, chemotherapies targeting known mechanisms have failed to lead to effective reversal of drug resistance, suggesting that alternative mechanisms remain undiscovered. Previous work identified midkine (MK) as a novel putative survival molecule responsible for cytoprotective signaling between drug-resistant and drug-sensitive neuroblastoma, osteosarcoma and breast carcinoma cells in vitro. In the present study, we provide further in vitro and in vivo studies supporting the role of MK in neuroblastoma cytoprotection. MK overexpressing wild type neuroblastoma cells exhibit a cytoprotective effect on wild type cells when grown in a co-culture system, similar to that seen with doxorubicin resistant cells. siRNA knockdown of MK expression in doxorubicin resistant neuroblastoma and osteosarcoma cells ameliorates this protective effect. Overexpression of MK in wild type neuroblastoma cells leads to acquired drug resistance to doxorubicin and to the related drug etoposide. Mouse studies injecting various ratios of doxorubicin resistant or MK transfected cells with GFP transfected wild type cells confirm this cytoprotective effect in vivo. These findings provide additional evidence for the existence of intercellular cytoprotective signals mediated by MK which contribute to chemotherapy resistance in neuroblastoma.

scholarly journals A UDP-GlcNAc : βGal β1-6 GlcNAc transferase involved in bloodstream form N-glycan and procyclic form GPI anchor elaboration in Trypanosoma brucei.

2021 ◽  
Author(s):  
Samuel Martin Duncan ◽  
Rupa Nagar ◽  
Manuela Damerow ◽  
Dmitry V. Yashunsky ◽  
Benedetta Buzzi ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Bloodstream Form ◽  
Wild Type ◽  
Dual Roles ◽  
Gpi Anchor ◽  
Procyclic Form ◽  
Life Cycle Stages ◽  
Glcnac Transferase

Trypanosoma brucei has large carbohydrate extensions on its N-linked glycans and glycosylphosphatidylinositol (GPI) anchors in its bloodstream form (BSF) and procyclic form (PCF), respectively. The parasites glycoconjugate repertoire suggests at least 38 glycosyltransferase (GT) activities, 16 of which are unknown. Here, we probe the function(s) of a putative β3GT gene, TbGT10. The BSF null mutant is viable in vitro and in vivo and can differentiate into PCF, demonstrating non-essentiality. However, the absence of TbGT10 led to impaired elaboration of N-glycans and GPI anchor sidechains in BSF and PCF parasites, respectively. Glycosylation defects include reduced BSF glycoprotein binding to ricin and to monoclonal antibodies mAb139 and mAbCB1. The latter bind a carbohydrate epitope of lysosomal glycoprotein p67 that we show here, using synthetic glycans, consists of (-6Gal1-4GlcNAc1-)≥4 poly-N-acetyllactosamine repeats. Methylation linkage analysis of Pronase glycopeptides isolated from BSF wild-type and TbGT10 null parasites show a reduction in 6-O-substituted- and 3,6-di-O-substituted-Gal residues. Together, these data suggest that TbGT10 encodes a UDP-GlcNAc : βGal β1-6 GlcNAc-transferase active in both BSF and PCF life-cycle stages elaborating complex N-glycans and GPI sidechains, respectively. The β1-6 specificity of this β3GT gene product and its dual roles in N-glycan and GPI glycan elaboration are notable.

Fas expression promotes proteasomal activity in toxin-induced parkinsonism

2012 ◽  
Vol 24 (3) ◽  
pp. 166-171
Author(s):  
Anne M. Landau ◽  
Rosmarie Siegrist-Johnstone ◽  
Julie Desbarats
Keyword(s):  
Neuroblastoma Cells ◽  
Death Receptor ◽  
Protein Aggregates ◽  
Ubiquitin Proteasome System ◽  
Wild Type ◽  
Proteasomal Activity ◽  
Ubiquitin Proteasome ◽  
Deficient Mice

Objective: Fas (CD95), commonly categorised as a death receptor due to its well-defined role in apoptosis, can paradoxically also promote neuroprotection. We have previously found that defects in Fas signalling render mice highly susceptible to neural degeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease (PD). Decreased activity of the ubiquitin proteasome system and accumulation of protein aggregates are implicated in PD pathogenesis. Here, we investigate the relationship between Fas and ubiquitin proteasomal activity in neuronal cells.Methods: We performed proteasome assays in neuroblastoma cells and in midbrain cultures of wild-type and Fas-deficient mice.Results: Neuroblastoma cells upregulated proteasomal activity in response to an activating Fas antibody in vitro. Furthermore, neural tissue from Fas-deficient mice showed decreased proteasomal activity compared with the tissue from wild-type mice when exposed to a PD-inducing toxin in vivo.Conclusion: These findings suggest that mechanisms for Fas-mediated neuroprotection may include Fas-induced upregulation of proteasomal activity, and consequently less accumulation of toxic protein aggregates.

scholarly journals Mutated but Not Deleted Ovine PrPCN-Terminal Polybasic Region Strongly Interferes with Prion Propagation in Transgenic Mice

2015 ◽  
Vol 90 (3) ◽  
pp. 1638-1646 ◽  
Author(s):  
Manal Khalifé ◽  
Fabienne Reine ◽  
Sophie Paquet-Fifield ◽  
Johan Castille ◽  
Laetitia Herzog ◽  
...  
Keyword(s):  
Amino Acid ◽  
Prion Protein ◽  
Major Effect ◽  
Cellular Prion Protein ◽  
Spongiform Encephalopathy ◽  
Structural Variations ◽  
Prion Propagation ◽  
Polybasic Region

ABSTRACTMammalian prions are proteinaceous infectious agents composed of misfolded assemblies of the host-encoded, cellular prion protein (PrP). Physiologically, the N-terminal polybasic region of residues 23 to 31 of PrP has been shown to be involved in its endocytic trafficking and interactions with glycosaminoglycans or putative ectodomains of membrane-associated proteins. Several recent reports also describe this PrP region as important for the toxicity of mutant prion proteins and the efficiency of prion propagation, bothin vitroandin vivo. The question remains as to whether the latter observations made with mouse PrP and mouse prions would be relevant to other PrP species/prion strain combinations given the dramatic impact on prion susceptibility of minimal amino acid substitutions and structural variations in PrP. Here, we report that transgenic mouse lines expressing ovine PrP with a deletion of residues 23 to 26 (KKRP) or mutated in this N-terminal region (KQHPH instead of KKRPK) exhibited a variable, strain-dependent susceptibility to prion infection with regard to the proportion of affected mice and disease tempo relative to findings in their wild-type counterparts. Deletion has no major effect on 127S scrapie prion pathogenesis, whereas mutation increased by almost 3-fold the survival time of the mice. Deletion marginally affected the incubation time of scrapie LA19K and ovine bovine spongiform encephalopathy (BSE) prions, whereas mutation caused apparent resistance to disease.IMPORTANCERecent reports suggested that the N-terminal polybasic region of the prion protein could be a therapeutic target to prevent prion propagation or toxic signaling associated with more common neurodegenerative diseases such as Alzheimer's disease. Mutating or deleting this region in ovine PrP completes the data previously obtained with the mouse protein by identifying the key amino acid residues involved.

scholarly journals Characterization of mesenchymal stem cells in sheep naturally infected with scrapie

2015 ◽  
Vol 96 (12) ◽  
pp. 3715-3726 ◽  
Author(s):  
Diego R. Mediano ◽  
David Sanz-Rubio ◽  
Rosa Bolea ◽  
Belén Marín ◽  
Francisco J. Vázquez ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Protein Misfolding ◽  
Prion Diseases ◽  
Transcript Level ◽  
Cellular Prion Protein ◽  
Jakob Disease

Mesenchymal stem cells (MSCs) can be infected with prions and have been proposed as in vitro cell-based models for prion replication. In addition, autologous MSCs are of interest for cell therapy in neurodegenerative diseases. To the best of our knowledge, the effect of prion diseases on the characteristics of these cells has never been investigated. Here, we analysed the properties of MSCs obtained from bone marrow (BM-MSCs) and peripheral blood (PB-MSCs) of sheep naturally infected with scrapie — a large mammal model for the study of prion diseases. After three passages of expansion, MSCs derived from scrapie animals displayed similar adipogenic, chondrogenic and osteogenic differentiation ability as cells from healthy controls, although a subtle decrease in the proliferation potential was observed. Exceptionally, mesenchymal markers such as CD29 were significantly upregulated at the transcript level compared with controls. Scrapie MSCs were able to transdifferentiate into neuron-like cells, but displayed lower levels of neurogenic markers at basal conditions, which could limit this potential. The expression levels of cellular prion protein (PrPC) were highly variable between cultures, and no significant differences were observed between control and scrapie-derived MSCs. However, during neurogenic differentiation the expression of PrPC was upregulated in MSCs. This characteristic could be useful for developing in vitro models for prion replication. Despite the infectivity reported for MSCs obtained from scrapie-infected mice and Creutzfeldt–Jakob disease patients, protein misfolding cyclic amplification did not detect PrPSc in BM- or PB-MSCs from scrapie-infected sheep, which limits their use for in vivo diagnosis for scrapie.

scholarly journals A tropane-based ibogaine analog rescues folding-deficient SERT and DAT

2020 ◽  
Author(s):  
Shreyas Bhat ◽  
Daryl A. Guthrie ◽  
Ameya Kasture ◽  
Ali El-Kasaby ◽  
Jianjing Cao ◽  
...  
Keyword(s):  
Protein Folding ◽  
Active Metabolite ◽  
Protein Misfolding ◽  
Ring System ◽  
Missense Mutations ◽  
Wild Type ◽  
Structural Requirements ◽  
Underlying Mechanisms

AbstractMissense mutations that give rise to protein misfolding are rare, but collectively, defective protein folding diseases are consequential. Folding deficiencies are amenable to pharmacological correction (pharmacochaperoning), but the underlying mechanisms remain enigmatic. Ibogaine and its active metabolite noribogaine correct folding defects in the dopamine transporter (DAT), but they rescue only a very limited number of folding-deficient DAT mutants, which give rise to infantile Parkinsonism and dystonia. Herein, a series of analogs was generated by reconfiguring the complex ibogaine ring system and exploring the structural requirements for binding to wild type transporters, and for rescuing two equivalent synthetic folding-deficient mutants, SERT-PG601,602AA and DAT-PG584,585AA. The most active tropane-based analog (9b) was also an effective pharmacochaperone in vivo, in Drosophila harboring DAT-PG584,585AA and rescued six out of 13 disease-associated human DAT mutants in vitro. Hence, a novel lead pharmacochaperone has been identified that demonstrates medication development potential for patients harboring DAT mutants.

Proteins P1, P2, and P0, components of the eukaryotic ribosome stalk. New structural and functional aspects

1995 ◽  
Vol 73 (11-12) ◽  
pp. 959-968 ◽  
Author(s):  
Miguel Remacha ◽  
Antonio Jimenez-Diaz ◽  
Cruz Santos ◽  
Elisa Briones ◽  
Reina Zambrano ◽  
...  
Keyword(s):  
Large Subunit ◽  
In Vitro Translation ◽  
Translation Regulation ◽  
Wild Type ◽  
Gtpase Domain ◽  
Two Dimensional Gel Electrophoresis ◽  
Ribosomal Stalk ◽  
Acidic Proteins

The eukaryoic ribosomal stalk is thought to consist of the phosphoproteins P1 and P2, which form a complex with protein P0. This complex interacts at the GTPase domain in the large subunit rRNA, overlapping the binding site of the protein L11-like eukaryotic counterpart (Saccharomyces cerevisiae protein L15 and mammalian protein LI2). An unusual pool of the dephosphorylated forms of proteins P1 and P2 is detected in eukaryotic cytoplasm, and an exchange between the proteins in the pool and on the ribosome takes place during translation. Quadruply disrupted yeast strains, carrying four inactive acidic protein genes and, therefore, containing ribosomes totally depleted of acidic proteins, are viable but grow with a doubling time threefold higher than wild-type cells. The in vitro translation systems derived from these stains are active but the two-dimensional gel electrophoresis pattern of proteins expressed in vivo and in vitro is partially different. These results indicate that the P1 and P2 proteins are not essential for ribosome activity but are able to affect the translation of some specific mRNAs. Protein P0 is analogous to bacterial ribosomal protein L10 but carries an additional carboxyl domain showing a high sequence homology to the acidic proteins P1 and P2, including the terminal peptide DDDMGFGLFD. Successive deletions of the P0 carboxyl domain show that removal of the last 21 amino acids from the P0 carboxyl domain only slightly affects the ribosome activity in a wild-type genetic background; however, the same deletion is lethal in a quadruple disruptant deprived of acidic P1/P2 proteins. Additional deletions affect the interaction of P0 with the P1 and P2 proteins and with the rRNA. The experimental data available support the implication of the eukaryotic stalk components in some regulatory process that modulates the ribosomal activity.Key words: ribosomal stalk, acidic proteins, phosphorylation, GTPase domain, translation regulation.

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