The Pathogenic A116V Mutation Enhances Ion-Selective Channel Formation by Prion Protein in Membranes
Authors: Jogender Singh
Ambadi Thody Sabareesan
Jayant B Udgaonkar
M K Mathew
Samrat Roy
Journal: Biophysical Journal (2016)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Prion diseases; PrP(Sc); transmembrane form; A116V mutation; Gerstmann-Sträussler-Scheinker syndrome
Affiliations: National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bengaluru, India., National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bengaluru, India; Biocon Bristol Myers Squibb Research Center, Bengaluru, India; School of Biotechnology, Kalinga Institute of Industrial Technology (KIIT) University, Bhubaneswar, India., National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bengaluru, India. Electronic address: .

Abstract:  Prion diseases are a group of fatal neurodegenerative disorders that afflict mammals. Misfolded and aggregated forms of the prion protein (PrP(Sc)) have been associated with many prion diseases. A transmembrane form of PrP favored by the pathogenic mutation A116V is associated with Gerstmann-Sträussler-Scheinker syndrome, but no accumulation of PrP(Sc) is detected. However, the role of the transmembrane form of PrP in pathological processes leading to neuronal death remains unclear. This study reports that the full-length mouse PrP (moPrP) significantly increases the permeability of living cells to K(+), and forms K(+)- and Ca(2+)-selective channels in lipid membranes. Importantly, the pathogenic mutation A116V greatly increases the channel-forming capability of moPrP. The channels thus formed are impermeable to sodium and chloride ions, and are blocked by blockers of voltage-gated ion channels. Hydrogen-deuterium exchange studies coupled with mass spectrometry (HDX-MS) show that upon interaction with lipid, the central hydrophobic region (109-132) of the protein is protected against exchange, making it a good candidate for inserting into the membrane and lining the channel. HDX-MS also shows a dramatic increase in the protein-lipid stoichiometry for A116V moPrP, providing a rationale for its increased channel-forming capability. The results suggest that ion channel formation may be a possible mechanism of PrP-mediated neurodegeneration by the transmembrane forms of PrP. 

Retracted: Yes

PubPeer comments:
 Sensitivity of tumour cells to methotrexate and the dependence on histidine was reported many years ago. Thus, this paper is not all that novel. I suggest the authors do a more thorough literature review in the future. https: www.ncbi.nlm.nih.gov pubmed 6883328 Selective killing of transformed cells by methotrexate with histidine deprivation or with alpha-amino alcohols. Newman EM, Nierenberg DW, Santi DV. Cancer Res. 1983 Oct;43(10):4703-8. Not cited in this paper by Sabatini s group. , 
 #1 Thank you for informing the scientific community on this unduly forgotten study. While I [am not surprised by the poor review of the prior literature here] , at least this paper brings these important findings back to the light and such a central news platform as *Nature* could inspire further scientific and medical work on them. Unfortunately, [the paper by Newman, Nierenberg Santi (1983)] became largely forgotten over the years and so far was cited only 8 times. Still, it would be nice on the part of the authors to add this important reference as a small correction. A recent addition of missed references by [Pastuzyn et al (2018) can serve as a nice example] . , 
 Supplemental Fig. 11 (a b) might contain image duplication. Could the authors check? 11a: ![file] 11b: ![file] , 
 #1 Thank you for your comment. Indeed, the title of the Newman et al. paper seems to suggest one of the conclusions of our work. However, the paper itself does not report any of the novel findings we made. The question that was addressed in the Newman et al. paper was whether growth arrest induced by amino acid deprivation results in reduced susceptibility to methotrexate. Histidine was used as an example of an essential amino acid whose deprivation results in growth arrest of 3T3 cells, but not of SV40-transformed 3T3 cells. Other amino acids that were tested and yielded similar results were tryptophan and tyrosine, even though their depletion from the growth media did not result in growth arrest, but rather slowed the cellsu2019 proliferation rate in roughly 50%. Newman et al. also studied the outcome of amino acid depletion by adding amino acid analogues to the media. Treatment with histidinol and tyrosynol also resulted in growth arrest and reduced sensitivity to methotrexate, but the toxic effects of these amino-alcohol supplements rendered the approach less favorable than amino acid depletion. In all these treatments the arrested 3T3 cells were less sensitive to methotrexate treatment due to a reduced need for nucleotide synthesis. No mechanism that is specific to histidine was reported, and in fact, it was emphasized by the authors that histidine deprivation is not unique in causing a growth arrest that protects cells from a cytotoxic drug: u201cAny of the conditions used which caused a plateau in the number of BALB 3T3 cells substantially protected the treated cells from cell death caused by MTX.u201d We, of course, also observed reduced sensitivity to methotrexate in histidine-deprived cells in culture, but we realized that these results were trivial and had been shown many times (by Newman et al. and many others since 1983), so we did not include these data in our manuscript. In summary, we did not cite the Newman et al paper because it discusses a phenomenon that we did not refer to in our manuscript (and in fact were careful to avoid). We trust that this response clarifies this issue and will prevent others from confusing the histidine-specific mechanism we found to protect cells from methotrexate toxicity from the general, well-established protective effect of nutrient deprivation and its concomitant reduction in the rate of cell proliferation. , 
 #3 Thank you for noticing this error. Indeed, we overlooked this mistake and we are happy to now correct it. Please see below for the corrected figure and we will also ask Nature if they wish to correct it on the online version of the paper. We emphasize that this mistake does not change the conclusion of the figure or any of the statements made in the paper. ![file] ![file] , 
 Could the authors please check Supplementary Figure 3? Two panels in the methotrexate column appear to look very similar. Marked with red boxes. It would be helpful if the authors could clarify if three rows of panels are supposed to show different mice or different sections of the same tissue sample. Each treatment group had 6 mice, so I would have expected that these photos would represent different mice. ![file] , 
 Raw data for Fig. 2 contains some duplications of values that are not expected. For example, TTP u2013 MTX and 5-methyl THF measurements provided the exact same values. ![file] The same is observed when you compare labeled TTP- untreated (RAMOS) with labeled TTP - MTX (HEL). Could the authors check, please? ![file] , 
 Another possible concern is the Survival MTX raw data. The assay performed in 04 19 17 (WT) got the same values as the experiments performed in 05 01 2017 for WT DOXO (2.5 to 25) ![file] Same can be verified observed for AAVS DOXO (04 19 17) is exactly the same values obtained in the assay performed in 05 01 2017 AAVS DOXO ![file] What is weirder is that the value for 0 is different but the value for the different concentrations are exactly the same, so it s just not an issue of copy paste of the whole assay, just part of it ![file] The table contains different other issues of exact values, mFTCD DOXO assay performed in 04 19 17 has the same values of the assay performed 05 01 2017 ![file] , 
 Other duplications of values spotted in the same table ![file] and ![file] Could the authors kindly explain what happened here? , 
 More similar than expected in the metabolomics raw data ![file] ![file] ![file] Could the authors check it please? , 
 #6 Response to Elisabeth M Bik Thank you for your comment. Indeed, I duplicated the top slide in the bottom row by mistake and will make sure to correct it. The corrected file is pasted below and we will contact the journal and correct the online version as well. This mistake does not change any of the conclusions of the paper. Additionally, I am happy to clarify that each H E capture is from a different mouse, and I apologize that it wasnu2019t clear before. Also, and in response to previous comments, a link to a dropbox folder with all the images I have from these mice is ready to be sent to whomever is interested. Please email me directly. I will be happy to comment and clarify any other issues you might have regarding the paper. Naama Kanarek ![file] , 
 #7 Dear Polypleurum Prachinburiense You are absolutely right! In my attempt to make the source data files clear I transposed each of the data sets and named the sets by cell line, treatment and metabolite. It was a mistake because it caused multiple errors. Below I pasted a screen shot of the data as it appears in my original file. In the file the top set refers to HEL cells and the bottom one to Ramos cells. Would you like me to send you this original file? It is quite easier to look at than the source data files.![file] , 
 #7 and here is the other: ![file] , 
 #10 Dear Epiplatys Zenkeri You are absolutely right regarding the IMP! Thank you very much for your attention to this. My original file is pated below as a screen shot. ![file] ![file] However, please let me explain what appears as repetition in the labeled 5,10-methenyl THF: We presented the data as fractions, so when one of the forms is not detected by the mass spec it appears as zero, and then the other form (M+1, labeled) is counted as 100% of the total 5,10-methenyl THF. This means that regardless of the value we measured in the M+1, if we couldn t detect any M+0 the value will always be 1. Since the total levels of folate in MTX-treated cells is low we sometimes couldn t detect both M+0 and M+1 forms. Every time this happened we got a 0 1 result. Please see the total values of 5,10-methenyl THF in the MTX-treated cells (pasted below) - they are really low, as expected in MTX-treated cells. ![file] , 
 I appreciate the efforts of the authors to clarify all the issues! However, could the authors also provide an explanation for the duplication of values in the viability data? (Commentary #8 and #9). , 
 #8 Response to comments 8 and 9: I thank the Epiplatys Zenkeri for detecting this error. Unlike the other errors indicated in the comments above, that were done only in the late versions of the manuscript and were very easy to detect and fix, this one traces back to my original data files and therefore required much more detective work on my partu2026 **I wish to emphasize that the error does not change the conclusion of the figure, and any of the statements in the paper.** The correction to the error is the number of biological repetitions for my control genotypes *in the control drug doxorubicin*: n=2 instead of n=3. Technical replicates for each biological replicate is still n=3. This does not change anything for the drug we studied u2013 methotrexate. I wish to explain the source of my error: In my analysis of survival data I used an excel template to calculate the dose response. I pasted the CellTiterGlo data from the plate reader into this pre-existing template. First, I paste the untreated values (replicate of n=6) into the untreated boxes of both MTX and DOXO tables (blue arrows in the figure below), then I paste the values of the different doses into the rest of the table. The template calculates the average value of the untreated wells (little orange arrows in the figure below), and then each of the treated wells is normalized to this value. On 05.01 I made a pipetting mistake of the CTG reagent and therefore excluded wells for doxo-treated WT 1uM and up and all the doxo-treated AAVS and mFTCD. Unfortunately, when I copied the data into my excel template it still had the results from the previous experiment and so I didnu2019t notice that the new pasted data got combined with the pre-existing one. I really regret this mistake. Obviously, simply by first deleting all pre-existing data I could have avoided that. I learned from this mistake and I sincerely hope to never repeat anything like this in the future. I am now working on my u201cAuthor correctionu201d that will be sent to Nature with the necessary corrections to my manuscript from the comments above. **I wish to emphasize that none of the errors change the conclusion of the figures, and any of the statements in the paper.** ![file] , 
 Author Correction: Histidine catabolism is a major determinant of methotrexate sensitivity https: www.nature.com articles s41586-021-03487-2 In Fig. 1f of this Article, one of the three biological replicates of the control drug doxorubicin was erroneously duplicated from another experiment. The original Fig. 1f is shown next to the corrected panel without the erroneous replicate as Fig. 1 of this Amendment. The Source Data for the original Fig. 1, which had included erroneous data in the tab u2018survival MTXu2019 regarding this doxorubicin replicate, has also been corrected and is shown as Supplementary Information to this Amendment. In the Fig. 1 legend, the text u201cu2026compared to wild-type cells (n = 3, biological replicates,u201d should have read: u2018u2026compared to wild-type cells (n = 3, biological replicates, except for doxorubicin in Ramos cells, where n = 2,u201d. In Extended Data Fig. 11 of this Article, two of the histological sections in the u2018methotrexateu2019 group (top and bottom images in Extended Data Fig. 11a) and two sections in the u2018vehicleu2019 group (middle and bottom images in Extended Data Fig. 11b) were duplicated, and the sections in the u2018histidine supplementationu2019 (bottom image in Fig. 11b) and u2018methotrexateu2019 (top image in Extended Data Fig. 11b) groups were also duplicated. The original panels of Extended Data Fig. 11 are shown next to the corrected panels as Fig. 2 of this Amendment. In Supplementary Fig. 3 of this Article, two of the histological sections in the u2018methotrexateu2019 group were duplicated. The original and corrected Supplementary Fig. 3 is shown as Supplementary Fig. 1 to this Amendment, with the new panels marked by red outlines. There were errors in the Source Data of Fig. 2 that have been corrected and have been highlighted in red in Supplementary Information to this Amendment; the data in the original Fig. 2 were correct. In the Methods section, the reference for the sgRNA library that was used for the screen (Wang et al. (2014), ref. 7) was incorrect, and the updated library was instead from Wang et al. (2015)1. The library can also be found here: http: www.addgene.org pooled-library sabatini-crispr-human-high-activity-3-sublibraries . These errors do not affect the conclusions of the paper, and the original Article has not been corrected online. David Sabatini is no longer affiliated with the Whitehead Institute or the Howard Hughes Medical Institute. At the request of the Whitehead Institute and to ensure execution of the duties of corresponding author, the corresponding author on this Author Correction is now Naama Kanarek (naama.kanarek@childrens.harvard.edu), replacing David Sabatini. All authors agree with the Author Correction and the change of the corresponding author. ,