Critical Significance of the Region between Helix 1 and 2 for Efficient Dominant-Negative Inhibition by Conversion-Incompetent Prion Protein

Taguchi, Yoshitaka; Mistica, Arla; Kitamoto, Tetsuyuki; Schätzl, Hermann

doi:10.1371/journal.ppat.1003466

Cited by 26 publications

(46 citation statements)

References 78 publications

(95 reference statements)

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“…Indeed although many inside deletions were done, so far they failed to generate prion entities still able to convert the wildtype PrP C . [10][11][12] We knew from our previous work that the sequence specificity of the C-terminal part of PrP helix H2 was not essential for prions, even if this sequence is highly conserved in mammalian PrP. Indeed, insertion of 8 extra amino acids in the last turns of the helix did not impair prion conversion.…”

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confidence: 99%

A stretch of residues within the protease-resistant core is not necessary for prion structure and infectivity

Muñoz-Montesino

Sizun

Moudjou

et al. 2017

Prion

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ABSTRACT. Mapping out regions of PrP influencing prion conversion remains a challenging issue complicated by the lack of prion structure. The portion of PrP associated with infectivity contains the a-helical domain of the correctly folded protein and turns into a b-sheet-rich insoluble core in prions. Deletions performed so far inside this segment essentially prevented the conversion. Recently we found that deletion of the last C-terminal residues of the helix H2 was fully compatible with prion conversion in the RK13-ovPrP cell culture model, using 3 different infecting strains. This was in agreement with preservation of the overall PrP C structure even after removal of up to one-third of this helix. Prions with internal deletion were infectious for cells and mice expressing the wild-type PrP and they retained prion strain-specific characteristics. We thus identified a piece of the prion domain that is neither necessary for the conformational transition of PrP C nor for the formation of a stable prion structure.

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confidence: 99%

A stretch of residues within the protease-resistant core is not necessary for prion structure and infectivity

Muñoz-Montesino

Sizun

Moudjou

et al. 2017

Prion

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“…To genetically delineate the critical determinants, a variety of deletions were introduced in the portion of PrP associated with infectivity, but thus far they were not compatible with prion conversion (23)(24)(25). Several deletions in PrP caused spontaneous neuropathologies in mice, but they were not associated with the production of PrP Sc or transmissibility (26)(27)(28)(29)(30)(31)(32).…”

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Generating Bona Fide Mammalian Prions with Internal Deletions

Muñoz-Montesino

Sizun

Moudjou

et al. 2016

J Virol

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Mammalian prions are PrP proteins with altered structures causing transmissible fatal neurodegenerative diseases. They are self-perpetuating through formation of beta-sheet-rich assemblies that seed conformational change of cellular PrP. Pathological PrP usually forms an insoluble protease-resistant core exhibiting beta-sheet structures but no more alpha-helical content, loosing the three alpha-helices contained in the correctly folded PrP. The lack of a high-resolution prion structure makes it difficult to understand the dynamics of conversion and to identify elements of the protein involved in this process. To determine whether completeness of residues within the protease-resistant domain is required for prions, we performed serial deletions in the helix H2 C terminus of ovine PrP, since this region has previously shown some tolerance to sequence changes without preventing prion replication. Deletions of either four or five residues essentially preserved the overall PrP structure and mutant PrP expressed in RK13 cells were efficiently converted into bona fide prions upon challenge by three different prion strains. Remarkably, deletions in PrP facilitated the replication of two strains that otherwise do not replicate in this cellular context. Prions with internal deletion were self-propagating and de novo infectious for naive homologous and wild-type PrP-expressing cells. Moreover, they caused transmissible spongiform encephalopathies in mice, with similar biochemical signatures and neuropathologies other than the original strains. Prion convertibility and transfer of strain-specific information are thus preserved despite shortening of an alpha-helix in PrP and removal of residues within prions. These findings provide new insights into sequence/structure/infectivity relationship for prions. IMPORTANCEPrions are misfolded PrP proteins that convert the normal protein into a replicate of their own abnormal form. They are responsible for invariably fatal neurodegenerative disorders. Other aggregation-prone proteins appear to have a prion-like mode of expansion in brains, such as in Alzheimer's or Parkinson's diseases. To date, the resolution of prion structure remains elusive. Thus, to genetically define the landscape of regions critical for prion conversion, we tested the effect of short deletions. We found that, surprisingly, removal of a portion of PrP, the C terminus of alpha-helix H2, did not hamper prion formation but generated infectious agents with an internal deletion that showed characteristics essentially similar to those of original infecting strains. Thus, we demonstrate that completeness of the residues inside prions is not necessary for maintaining infectivity and the main strain-specific information, while reporting one of the few if not the only bona fide prions with an internal deletion.

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“…36%, 33% and finally homogenization buffer (8.5%), 1.5 ml each, from bottom to top (Taguchi et al , 2013). …”

Section: Methodsmentioning

confidence: 99%

“… Here, we introduce the protocol for small-scale and simple subcellular fractionation used in our recent publication (Taguchi et al , 2013), which uses homogenization by passing through needles and sucrose step-gradient. Subcellular fractionation is a very useful technique but usually a large number of cells are required. Because we needed subcellular fractionation of transiently-transfected cells, we developed a protocol for smaller numbers of cells.…”

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Small-scale Subcellular Fractionation with Sucrose Step Gradient

Taguchi

Schätzl

2014

BIO-PROTOCOL

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Here, we introduce the protocol for small-scale and simple subcellular fractionation used in our recent publication (Taguchi et al., 2013), which uses homogenization by passing through needles and sucrose step-gradient. Subcellular fractionation is a very useful technique but usually a large number of cells are required. Because we needed subcellular fractionation of transiently-transfected cells, we developed a protocol for smaller numbers of cells. Our protocol for the subcellular fractionation is based on the protocol published by de Araújo and Huber (de Araujo et al., 2007), although substantial modifications have been made according to our experiences and information from personal communications. As optimal conditions seem to vary between cell lines, we advise to further modify the protocol to optimize for individual experiments. Our method is simple but sufficient for analysis of integral membrane proteins or proteins anchored to organelles by glycosylphosphatidylinositol or other lipid anchors, e.g. prion protein. However, proteins non-covalently attached to membranes or membrane proteins of organelles seem to be more prone to dissociation from the organelles during preparation and, if these proteins are the object of study, further modifications might be necessary. Unlike in a continuous gradient, where a protein of interest is scattered over a wide range, step-gradient fractionation is advantageous in detection of relatively small amounts of proteins from small-scale experiments, because it concentrates the protein of interest in one fraction, if an appropriate combination of sucrose concentrations is used.

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Critical Significance of the Region between Helix 1 and 2 for Efficient Dominant-Negative Inhibition by Conversion-Incompetent Prion Protein

Cited by 26 publications

References 78 publications

A stretch of residues within the protease-resistant core is not necessary for prion structure and infectivity

A stretch of residues within the protease-resistant core is not necessary for prion structure and infectivity

Generating Bona Fide Mammalian Prions with Internal Deletions

Small-scale Subcellular Fractionation with Sucrose Step Gradient

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