Critical Significance of the Region between Helix 1 and 2 for Efficient Dominant-Negative Inhibition by Conversion-Incompetent Prion Protein
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{"title"=>"Critical Significance of the Region between Helix 1 and 2 for Efficient Dominant-Negative Inhibition by Conversion-Incompetent Prion Protein", "type"=>"journal", "authors"=>[{"first_name"=>"Yuzuru", "last_name"=>"Taguchi", "scopus_author_id"=>"7201865827"}, {"first_name"=>"Arla M A", "last_name"=>"Mistica", "scopus_author_id"=>"56049906400"}, {"first_name"=>"Tetsuyuki", "last_name"=>"Kitamoto", "scopus_author_id"=>"7101608160"}, {"first_name"=>"Hermann M.", "last_name"=>"Schätzl", "scopus_author_id"=>"7004486352"}], "year"=>2013, "source"=>"PLoS Pathogens", "identifiers"=>{"pui"=>"369208246", "sgr"=>"84879544713", "pmid"=>"23825952", "scopus"=>"2-s2.0-84879544713", "isbn"=>"1553-7374 (Electronic)\\r1553-7366 (Linking)", "doi"=>"10.1371/journal.ppat.1003466", "issn"=>"15537366"}, "id"=>"6d1ef571-060d-3f2a-ab17-cec89da159a6", "abstract"=>"Prion diseases are fatal infectious neurodegenerative disorders in man and animals associated with the accumulation of the pathogenic isoform PrP(Sc) of the host-encoded prion protein (PrP(c)). A profound conformational change of PrP(c) underlies formation of PrP(Sc) and prion propagation involves conversion of PrP(c) substrate by direct interaction with PrP(Sc) template. Identifying the interfaces and modalities of inter-molecular interactions of PrPs will highly advance our understanding of prion propagation in particular and of prion-like mechanisms in general. To identify the region critical for inter-molecular interactions of PrP, we exploited here dominant-negative inhibition (DNI) effects of conversion-incompetent, internally-deleted PrP (ΔPrP) on co-expressed conversion-competent PrP. We created a series of ΔPrPs with different lengths of deletions in the region between first and second α-helix (H1∼H2) which was recently postulated to be of importance in prion species barrier and PrP fibril formation. As previously reported, ΔPrPs uniformly exhibited aberrant properties including detergent insolubility, limited protease digestion resistance, high-mannose type N-linked glycans, and intracellular localization. Although formerly controversial, we demonstrate here that ΔPrPs have a GPI anchor attached. Surprisingly, despite very similar biochemical and cell-biological properties, DNI efficiencies of ΔPrPs varied significantly, dependant on location and inversely correlated with the size of deletion. This data demonstrates that H1∼H2 and the region C-terminal to it are critically important for efficient DNI. It also suggests that this region is involved in PrP-PrP interaction and conversion of PrP(C) into PrP(Sc). To reconcile the paradox of how an intracellular PrP can exert DNI, we demonstrate that ΔPrPs are subject to both proteasomal and lysosomal/autophagic degradation pathways. Using autophagy pathways ΔPrPs obtain access to the locale of prion conversion and PrP(Sc) recycling and can exert DNI there. This shows that the intracellular trafficking of PrPs is more complex than previously anticipated.", "link"=>"http://www.mendeley.com/research/critical-significance-region-between-helix-1-2-efficient-dominantnegative-inhibition-conversionincom", "reader_count"=>24, "reader_count_by_academic_status"=>{"Researcher"=>6, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>10, "Student > Master"=>4, "Other"=>1, "Professor"=>2}, "reader_count_by_user_role"=>{"Researcher"=>6, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>10, "Student > Master"=>4, "Other"=>1, "Professor"=>2}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>7, "Agricultural and Biological Sciences"=>14, "Medicine and Dentistry"=>1, "Veterinary Science and Veterinary Medicine"=>2}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>14}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>7}, "Veterinary Science and Veterinary Medicine"=>{"Veterinary Science and Veterinary Medicine"=>2}}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1103484"], "description"=>"<p><b>A.</b> ΔPrPs are insoluble in detergent solution. Immunoblots showing partition of Δ159 and Δ159–175 in supernatant (Sup) and pellet fraction after ultracentrifugation of lysates supplemented with 4% sarcosyl. Upper panel is mAb 3F4, lower panel is membrane re-probed with mAb 4H11 detecting also endogenous wild-type PrP besides ΔPrPs. The majority of ΔPrPs is found in the insoluble fraction in both infected and non–infected cells (upper panel, lanes 3, 4, 7 and 8). Wild-type PrP almost completely partitions into the soluble fraction (Sup) in non-infected N2a cells with present conditions. PrP in pellet fractions of non-infected N2a cells is from ΔPrPs as can be seen from the banding pattern (Lane 7 and 8, square bracket). PrP in lanes 3 and 4 (lower panel) is PrP27-30. <b>B.</b> ΔPrPs are moderately resistant to chymotrypsin digestion. Immunoblots showing chymotrypsin-resistant fragments of Δ159 and Δ159–175 from transiently transfected N2a cell lysates digested with indicated concentrations of chymotrypsin (Chy). Note that full-length diglycoform of ΔPrP still remains at 8 µg/ml Chy (lanes 5 and 10, upper panel, closed arrowhead), whereas that of endogenous wild-type PrP is completely digested (lanes 5 and 10, lower panel, open arrowhead) and only small amounts of truncated fragments remained (curly bracket). Other smaller fragments in lane 5 and 10 (square-brackets) represent ΔPrPs.</p>", "links"=>[], "tags"=>["Biochemistry", "glycobiology", "glycoproteins", "metabolism", "Protein metabolism", "proteins", "Protein interactions", "protein structure", "Recombinant proteins", "biophysics", "Protein folding", "Infectious diseases", "Prion diseases", "Veterinary diseases", "Zoonotic diseases", "Veterinary prion diseases", "spontaneously", "aggregates", "detergent-insoluble"], "article_id"=>733385, "categories"=>["Medicine", "Biological Sciences"], "users"=>["Yuzuru Taguchi", "Arla M. A. Mistica", "Tetsuyuki Kitamoto", "Hermann M. Schätzl"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1003466.g003", "stats"=>{"downloads"=>0, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_916_PrPs_spontaneously_form_aggregates_that_are_detergent_insoluble_and_relatively_chymotrypsin_resistant_/733385", "title"=>"ΔPrPs spontaneously form aggregates that are detergent-insoluble and relatively chymotrypsin-resistant.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-06-27 02:51:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1103496"], "description"=>"<p><b>A.</b> DNI efficiencies of ΔPrPs are inversely correlated to deletion size. Representative immunoblot showing PK-resistant PrP<sup>Sc</sup> moiety of (3F4)MoPrP co-expressed with indicated ΔPrPs in 22L-ScN2a cells. Quantitative analysis is shown below as scheme. Relative PrP<sup>Sc</sup> levels were calculated as proportion of PK-resistant (3F4)MoPrP co-transfected with each ΔPrP to that of co-transfected with empty vector control in the same experiment. Data from 4 (for Δ159–165, Δ159–167 and Δ159–167(169) only 3) independent experiments were statistically analyzed for mean and standard deviation (error bars). <b>B.</b> DNI of ΔPrP with a deletion in the C-terminal part of H1∼H2. Δ171–175 (see Fig. 7A) was transiently expressed in N2a cells (left panel) and DNI efficiency evaluated in 22L-ScN2a cells (right panel) (mAb 3F4). Despite the much shorter deletion size, Δ171–175 exerted only inefficient DNI, similar to Δ159–175 (right panel).</p>", "links"=>[], "tags"=>["Biochemistry", "glycobiology", "glycoproteins", "metabolism", "Protein metabolism", "proteins", "Protein interactions", "protein structure", "Recombinant proteins", "biophysics", "Protein folding", "Infectious diseases", "Prion diseases", "Veterinary diseases", "Zoonotic diseases", "Veterinary prion diseases", "dni", "deletion", "positioning"], "article_id"=>733397, "categories"=>["Medicine", "Biological Sciences"], "users"=>["Yuzuru Taguchi", "Arla M. A. Mistica", "Tetsuyuki Kitamoto", "Hermann M. Schätzl"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1003466.g006", "stats"=>{"downloads"=>0, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Inverse_correlation_of_DNI_and_size_of_deletion_and_importance_of_positioning_of_deletion_/733397", "title"=>"Inverse correlation of DNI and size of deletion and importance of positioning of deletion.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-06-27 02:51:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1103477"], "description"=>"<p><b>A.</b> Schematic illustration of prion protein secondary structure elements comprising two β-strands (B1 and B2) and three α-helices (H1–3) and post-translational modifications (two N-linked glycans and glycosylphosphatidylinositol anchor) along with position and extent of deletion of ΔPrPs constructs. Deletions in the H1∼H2 portion have a common N-terminal end (residue 159) and gradually extend into the C-terminal direction. All constructs have a 3F4 epitope tag (methionines at residue 108 and 111). Underlined residues denote B1. <b>B.</b> Comparison of ΔPrP expression levels. Representative immunoblot is shown for detection of (3F4)MoPrP and ΔPrPs in transiently transfected N2a cells using mAb 3F4. ΔPrPs are similar in expression level glycosylation appearance, except for Δ159–167 which has an extra fragment larger than the diglycoform (lane 7, arrow). <b>C.</b> ΔPrPs are not converted into PK-resistant PrP in prion-infected cells. 22L-ScN2a cells were transiently transfected with (3F4)MoPrP, Δ159 or Δ159–175, and cell lysates digested with PK or not. Immunoblot was done using mAb 3F4. <b>D.</b> Pilot study for dominant-negative inhibition (DNI) of Δ159 and Δ159–175. DNI was assessed by co-transfecting 22L-ScN2a cells with (3F4)MoPrP and Δ159 or Δ159–175, respectively, and testing for PK-resistant (3F4)MoPrP. Left panel shows representative immunoblot (mAb 3F4) and right panel the statistical analysis of quantified PK-res levels of a triplicate experiment. Empty pcDNA3.1 plasmid was used as control for co-transfection in lane 1. The error bars indicate standard deviation. *, p<0.05.</p>", "links"=>[], "tags"=>["Biochemistry", "glycobiology", "glycoproteins", "metabolism", "Protein metabolism", "proteins", "Protein interactions", "protein structure", "Recombinant proteins", "biophysics", "Protein folding", "Infectious diseases", "Prion diseases", "Veterinary diseases", "Zoonotic diseases", "Veterinary prion diseases", "dominant-negative", "inhibition", "persistently", "prion-infected"], "article_id"=>733378, "categories"=>["Medicine", "Biological Sciences"], "users"=>["Yuzuru Taguchi", "Arla M. A. Mistica", "Tetsuyuki Kitamoto", "Hermann M. Schätzl"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1003466.g001", "stats"=>{"downloads"=>2, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_of_916_PrPs_and_dominant_negative_inhibition_DNI_of_916_159_and_916_159_8211_175_in_persistently_prion_infected_cells_/733378", "title"=>"Expression of ΔPrPs and dominant-negative inhibition (DNI) of Δ159 and Δ159–175 in persistently prion-infected cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-06-27 02:51:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1103490"], "description"=>"<p><b>A.</b> ΔPrPs are substantially soluble in detergent without GdnHCl denaturation. Immunoblots probed with mAbs 3F4 (left) or 4H11 (right, membrane re-probed) showing ΔPrPs and endogenous PrP in detergent and aqueous phases of TX114 lysates prepared from N2a cells transfected with Δ159 or Δ159–175. Det denotes detergent phase, Aq aqueous phase. <b>B.</b> ΔPrPs are highly hydrophobic after denaturation with GdnHCl. Representative immunoblot of TX114-treated lysates after denaturation with 3 M GdnHCl and developed with mAb 3F4 showing Δ159 and Δ159–175 almost exclusively in detergent phase (lanes 1–4). PIPLC treatment (+, even lanes) was done after denaturation with GdnHCl or not (−, uneven lanes). PIPLC treatment did not significantly influence ΔPrP partitioning under these conditions. <b>C.</b> Hydrophobicity of ΔPrPs combined with sensitivity to PIPLC digestion. After further diluting out GdnHCl by phase-separation and removal of the aqueous phase containing GdnHCl, PIPLC was able to digest ΔPrPs. Left two panels (upper mAb 3F4, lower 4H11) are shorter expositions, right panels longer expositions to show also difference in PrP levels in the detergent phase. Under these conditions a substantial reduction of ΔPrPs in the detergent phase was achieved by PIPLC digestion (e.g. upper-left panel, lanes 1, 3 vs. 2, 4). Migration of ΔPrPs in the aqueous phase of PIPLC-digested samples was slightly slower than that of PIPLC-undigested samples (−) (upper-right panel, lane 9 vs. 10).</p>", "links"=>[], "tags"=>["Biochemistry", "glycobiology", "glycoproteins", "metabolism", "Protein metabolism", "proteins", "Protein interactions", "protein structure", "Recombinant proteins", "biophysics", "Protein folding", "Infectious diseases", "Prion diseases", "Veterinary diseases", "Zoonotic diseases", "Veterinary prion diseases", "gpi-anchor"], "article_id"=>733391, "categories"=>["Medicine", "Biological Sciences"], "users"=>["Yuzuru Taguchi", "Arla M. A. Mistica", "Tetsuyuki Kitamoto", "Hermann M. Schätzl"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1003466.g004", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_916_PrPs_have_a_GPI_anchor_attached_/733391", "title"=>"ΔPrPs have a GPI-anchor attached.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-06-27 02:51:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1103499"], "description"=>"<p><b>A.</b> Schematic illustration of Δ171–175, Δ31–160 and its variants. Δ31–160 variants lack the entire part between the polybasic motif (PB, italic and bold) and residue 161. SS, signal sequence. <b>B.</b> Δ31–160 variants are expressed at comparable levels and have EndoH-sensitive and –resistant N-glycans. Immunoblot comparing EndoH-digested and non-digested samples from N2a cells transiently transfected with Δ31–160 variants, probed with anti-PrP mAb C16-S raised against the C-terminal portion of H3. Non-Tf, samples prepared from N2a cells without transfection. Arrowhead denotes deglycosylated Δ159 (lane 11), bracket deglycosylated fragments, square bracket EndoH-resistant fragments. Asterisks indicate non-specific bands. Δ31–160 variants are not completely deglycosylated by EndoH, resulting in EndoH-resistant fragments (square bracket) which are observed also in reducing conditions (right panel; +/− dithiothreitol (DTT) treatment). Note that endogenous wild-type PrP<sup>c</sup> was not detected under used conditions. <b>C.</b> DNI of Δ31–160 variants is similarly inversely related to size of deletion and dependent on intact C-terminal H1–H2 portion. Immunoblot probed with mAb 3F4 showing PK-resistant (3F4)MoPrP co-transfected with indicated constructs into 22L-ScN2a cells (left panel). Δ31–160 was slightly more effective than Δ159. Right panel shows quantification of results as obtained from a triplicate experiment, using densitometry on ImageJ. Bars illustrate mean ± standard deviation. *, p<0.05. <b>D.</b> Δ31–160 forms spontaneous aggregates with moderate chymotrypsin (Chy) resistance. Immunoblots probed with mAbs C16-S (upper panel) or 3F4 (lower panel) for comparing Chy-resistant fragments of Δ31–160 with that of Δ159. Lysates from N2a cells expressing Δ159 or Δ31–160 were digested with indicated concentrations of Chy for 30 minutes and then digested with PNGaseF. A sample from non-transfected N2a cells is shown in the lane 5. Asterisks indicate non-specific C16-S bands. Arrowhead denotes deglycoform of Δ159, bracket deglycoform of Δ31–160.</p>", "links"=>[], "tags"=>["Biochemistry", "glycobiology", "glycoproteins", "metabolism", "Protein metabolism", "proteins", "Protein interactions", "protein structure", "Recombinant proteins", "biophysics", "Protein folding", "Infectious diseases", "Prion diseases", "Veterinary diseases", "Zoonotic diseases", "Veterinary prion diseases", "lacking", "n-terminal", "exert"], "article_id"=>733400, "categories"=>["Medicine", "Biological Sciences"], "users"=>["Yuzuru Taguchi", "Arla M. A. Mistica", "Tetsuyuki Kitamoto", "Hermann M. Schätzl"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1003466.g007", "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_916_PrPs_lacking_in_addition_the_N_terminal_region_still_exert_efficient_DNI_/733400", "title"=>"ΔPrPs lacking in addition the N-terminal region still exert efficient DNI.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-06-27 02:51:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1103480"], "description"=>"<p><b>A.</b> N-linked glycans of ΔPrPs are not complex-type. Immunoblot comparing EndoH- and non-digested lysates of ΔPrP159 and Δ159–175 transfected cells. Left panel was probed with mAb 3F4 mAb, detecting transiently expressed ΔPrPs. On the right panel, the membrane was re-probed with mAb 4H11, detecting total PrP. Arrowheads depict deglycosylated ΔPrPs. <b>B.</b> The additional band of Δ159–167 represents a third N-glycan sensitive to EndoH. Upon EndoH digestion, all bands converge into a non-glycoform band (lane 3 and 4). In Δ159–167(169) the third glycosylation site is not present anymore. <b>C.</b> ΔPrPs are mainly distributed in intracellular compartments rather than on the cell surface. Confocal microscopy images of N2a cells transiently transfected with (3F4)MoPrP, Δ159 or Δ159–175, either permeabilized (right) or non-permeabilized (left) with 0.2% TX100 after fixation. Cells were immuno-labeled with mAb 3F4. (3F4)MoPrP-expressing cells show bright immunopositivity on the cell surface, whereas ΔPrPs are not observed on the cell surface. Also intracellular staining differs strongly between (3F4)MoPrP and ΔPrP expressing cells Scale bars, 25 µm.</p>", "links"=>[], "tags"=>["Biochemistry", "glycobiology", "glycoproteins", "metabolism", "Protein metabolism", "proteins", "Protein interactions", "protein structure", "Recombinant proteins", "biophysics", "Protein folding", "Infectious diseases", "Prion diseases", "Veterinary diseases", "Zoonotic diseases", "Veterinary prion diseases", "non-complex", "glycosylated", "retained"], "article_id"=>733381, "categories"=>["Medicine", "Biological Sciences"], "users"=>["Yuzuru Taguchi", "Arla M. A. Mistica", "Tetsuyuki Kitamoto", "Hermann M. Schätzl"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1003466.g002", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_916_PrPs_are_non_complex_glycosylated_and_retained_intracellularly_/733381", "title"=>"ΔPrPs are non-complex glycosylated and retained intracellularly.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-06-27 02:51:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1103494"], "description"=>"<p><b>A.</b> Degradation of ΔPrPs by lysosomal and proteasomal systems. (Left) N2a cells transiently transfected with Δ159 or Δ159–175 were treated for 7 hours with inhibitors either for lysosomal degradation (bafilomycin A1, Baf, 120 nM), autophagy (3-methyladenine, 3MA, 10 mM), or the proteasome (MG132, 10 µM) and cell lysates analyzed in immunoblot for expression of Δ159 and Δ159–175 (mAb 3F4). Square bracket and bracket denote diglycoforms and nonglycoforms of ΔPrPs, respectively. An increment in diglycoforms is observed in Baf- and 3MA-treated cells, whereas in MG132-treated cells also the non-glycoforms are increased. (Right) A graph showing quantification of all PrP bands by densitometric analysis. Data from 3 independent (one in duplicate) experiments for Δ159 and 2 independent (one in duplicate) for Δ159–175 were statistically analyzed for mean and standard deviation (error bars). <b>B.</b> ΔPrPs can be localized in LAMP1-immunopositive vesicles. Immunofluorescence analysis shows distribution of ΔPrP (mAb 3F4) and LAMP1 in cells with or without 6M GdnHCl antigen-retrieval treatment. N2a cells transfected with ΔPrP159 were fixed, permeabilized and incubated without (upper panel) or with 6M GdnHCl (lower panel). With GndHCl treatment ΔPrP is diffusely distributed (arrowheads) and co-localizes poorly with LAMP1. After GdnHCl treatment (lower panel), bright 3F4-immunopositive puncta (arrows) are observed which partly co-localize with LAMP1-positive structures (merge). Scale bars, 10 µm.</p>", "links"=>[], "tags"=>["Biochemistry", "glycobiology", "glycoproteins", "metabolism", "Protein metabolism", "proteins", "Protein interactions", "protein structure", "Recombinant proteins", "biophysics", "Protein folding", "Infectious diseases", "Prion diseases", "Veterinary diseases", "Zoonotic diseases", "Veterinary prion diseases", "degraded", "lysosomal", "proteasomal", "degradation"], "article_id"=>733395, "categories"=>["Medicine", "Biological Sciences"], "users"=>["Yuzuru Taguchi", "Arla M. A. Mistica", "Tetsuyuki Kitamoto", "Hermann M. Schätzl"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1003466.g005", "stats"=>{"downloads"=>0, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_916_PrPs_are_degraded_by_both_lysosomal_and_proteasomal_degradation_systems_/733395", "title"=>"ΔPrPs are degraded by both lysosomal and proteasomal degradation systems.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-06-27 02:51:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1103511", "https://ndownloader.figshare.com/files/1103512", "https://ndownloader.figshare.com/files/1103513", "https://ndownloader.figshare.com/files/1103514", "https://ndownloader.figshare.com/files/1103515", "https://ndownloader.figshare.com/files/1103517"], "description"=>"<div><p>Prion diseases are fatal infectious neurodegenerative disorders in man and animals associated with the accumulation of the pathogenic isoform PrP<sup>Sc</sup> of the host-encoded prion protein (PrP<sup>c</sup>). A profound conformational change of PrP<sup>c</sup> underlies formation of PrP<sup>Sc</sup> and prion propagation involves conversion of PrP<sup>c</sup> substrate by direct interaction with PrP<sup>Sc</sup> template. Identifying the interfaces and modalities of inter-molecular interactions of PrPs will highly advance our understanding of prion propagation in particular and of prion-like mechanisms in general. To identify the region critical for inter-molecular interactions of PrP, we exploited here dominant-negative inhibition (DNI) effects of conversion-incompetent, internally-deleted PrP (ΔPrP) on co-expressed conversion-competent PrP. We created a series of ΔPrPs with different lengths of deletions in the region between first and second α-helix (H1∼H2) which was recently postulated to be of importance in prion species barrier and PrP fibril formation. As previously reported, ΔPrPs uniformly exhibited aberrant properties including detergent insolubility, limited protease digestion resistance, high-mannose type N-linked glycans, and intracellular localization. Although formerly controversial, we demonstrate here that ΔPrPs have a GPI anchor attached. Surprisingly, despite very similar biochemical and cell-biological properties, DNI efficiencies of ΔPrPs varied significantly, dependant on location and inversely correlated with the size of deletion. This data demonstrates that H1∼H2 and the region C-terminal to it are critically important for efficient DNI. It also suggests that this region is involved in PrP-PrP interaction and conversion of PrP<sup>C</sup> into PrP<sup>Sc</sup>. To reconcile the paradox of how an intracellular PrP can exert DNI, we demonstrate that ΔPrPs are subject to both proteasomal and lysosomal/autophagic degradation pathways. Using autophagy pathways ΔPrPs obtain access to the locale of prion conversion and PrP<sup>Sc</sup> recycling and can exert DNI there. This shows that the intracellular trafficking of PrPs is more complex than previously anticipated.</p></div>", "links"=>[], "tags"=>["Biochemistry", "glycobiology", "glycoproteins", "metabolism", "Protein metabolism", "proteins", "Protein interactions", "protein structure", "Recombinant proteins", "biophysics", "Protein folding", "Infectious diseases", "Prion diseases", "Veterinary diseases", "Zoonotic diseases", "Veterinary prion diseases", "helix", "dominant-negative", "inhibition", "conversion-incompetent", "prion"], "article_id"=>733407, "categories"=>["Medicine", "Biological Sciences"], "users"=>["Yuzuru Taguchi", "Arla M. A. Mistica", "Tetsuyuki Kitamoto", "Hermann M. Schätzl"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1003466.s001", "https://dx.doi.org/10.1371/journal.ppat.1003466.s002", "https://dx.doi.org/10.1371/journal.ppat.1003466.s003", "https://dx.doi.org/10.1371/journal.ppat.1003466.s004", "https://dx.doi.org/10.1371/journal.ppat.1003466.s005", "https://dx.doi.org/10.1371/journal.ppat.1003466.s006"], "stats"=>{"downloads"=>1, "page_views"=>53, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Critical_Significance_of_the_Region_between_Helix_1_and_2_for_Efficient_Dominant_Negative_Inhibition_by_Conversion_Incompetent_Prion_Protein_/733407", "title"=>"Critical Significance of the Region between Helix 1 and 2 for Efficient Dominant-Negative Inhibition by Conversion-Incompetent Prion Protein", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-06-27 02:51:42"}

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Relative Metric

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