Structural and Functional Characterization of the Most Frequent Pathogenic PRKN Substitution p.R275W

Mutations in the PINK1 and PRKN genes are the most frequent genetic cause of early-onset Parkinson disease. The pathogenic p.R275W substitution in PRKN is the most frequent substitution observed in patients, and thus far has been characterized mostly through overexpression models that suggest a poss...

Full description

Saved in:
Bibliographic Details
Published in:Cells (Basel, Switzerland) Switzerland), 2024-09, Vol.13 (18), p.1540
Main Authors: Bustillos, Bernardo A., Cocker, Liam T., Coban, Mathew A., Weber, Caleb A., Bredenberg, Jenny M., Boneski, Paige K., Siuda, Joanna, Slawek, Jaroslaw, Puschmann, Andreas, Narendra, Derek P., Graff-Radford, Neill R., Wszolek, Zbigniew K., Dickson, Dennis W., Ross, Owen A., Caulfield, Thomas R., Springer, Wolfdieter, Fiesel, Fabienne C.
Format: Article
Language:English
Citations: Items that this one cites
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
cited_by
cites cdi_FETCH-LOGICAL-c770-255c1a2a2775ae6182c83184592dd2b3520ed4f68d472cd04c183af9e30653ab3
container_end_page
container_issue 18
container_start_page 1540
container_title Cells (Basel, Switzerland)
container_volume 13
creator Bustillos, Bernardo A.
Cocker, Liam T.
Coban, Mathew A.
Weber, Caleb A.
Bredenberg, Jenny M.
Boneski, Paige K.
Siuda, Joanna
Slawek, Jaroslaw
Puschmann, Andreas
Narendra, Derek P.
Graff-Radford, Neill R.
Wszolek, Zbigniew K.
Dickson, Dennis W.
Ross, Owen A.
Caulfield, Thomas R.
Springer, Wolfdieter
Fiesel, Fabienne C.
description Mutations in the PINK1 and PRKN genes are the most frequent genetic cause of early-onset Parkinson disease. The pathogenic p.R275W substitution in PRKN is the most frequent substitution observed in patients, and thus far has been characterized mostly through overexpression models that suggest a possible gain of toxic misfunction. However, its effects under endogenous conditions are largely unknown. We used patient fibroblasts, isogenic neurons, and post-mortem human brain samples from carriers with and without PRKN p.R275W to assess functional impact. Immunoblot analysis and immunofluorescence were used to study mitophagy activation, and mitophagy execution was analyzed by flow cytometry of the reporter mitoKeima. The functional analysis was accompanied by structural investigation of PRKN p.R275W. We observed lower PRKN protein in fibroblasts with compound heterozygous p.R275W mutations. Isogenic neurons showed an allele-dose dependent decrease in PRKN protein. Lower PRKN protein levels were accompanied by diminished phosphorylated ubiquitin and decreased MFN2 modification. Mitochondrial degradation was also allele-dose dependently impaired. Consistently, PRKN protein levels were drastically reduced in human brain samples from p.R275W carriers. Finally, structural simulations showed significant changes in the closed form of PRKN p.R275W. Our data suggest that under endogenous conditions the p.R275W mutation results in a loss-of-function by destabilizing PRKN.
doi_str_mv 10.3390/cells13181540
format article
fullrecord <record><control><sourceid>crossref</sourceid><recordid>TN_cdi_crossref_primary_10_3390_cells13181540</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>10_3390_cells13181540</sourcerecordid><originalsourceid>FETCH-LOGICAL-c770-255c1a2a2775ae6182c83184592dd2b3520ed4f68d472cd04c183af9e30653ab3</originalsourceid><addsrcrecordid>eNpVUMtOwzAQtBBIVKVH7v6BFD9j54giAogCVVuJY7SxHRIUkmI7B_j6psAB5jIPaVbaQeiSkiXnGbkyrusC5VRTKcgJmjGieCIEyU7_6HO0COGNTNA0pUTOkN1GP5o4eugw9BYXY29iO_STzRvwYKLz7RccIzzUODYOPw4h4sK7j9H1Ea8hNsOr61uD15uHJ7wdqxDbOH439ssNU_LlAp3V0AW3-OU52hU3u_wuWT3f3ufXq8QoRRImpaHAgCklwaVUM6Onf4TMmLWs4pIRZ0WdaisUM5YIQzWHOnOcpJJDxeco-Tlr_BCCd3W59-07-M-SkvI4UvlvJH4AAiJazQ</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype></control><display><type>article</type><title>Structural and Functional Characterization of the Most Frequent Pathogenic PRKN Substitution p.R275W</title><source>PubMed (Medline)</source><source>Publicly Available Content Database</source><creator>Bustillos, Bernardo A. ; Cocker, Liam T. ; Coban, Mathew A. ; Weber, Caleb A. ; Bredenberg, Jenny M. ; Boneski, Paige K. ; Siuda, Joanna ; Slawek, Jaroslaw ; Puschmann, Andreas ; Narendra, Derek P. ; Graff-Radford, Neill R. ; Wszolek, Zbigniew K. ; Dickson, Dennis W. ; Ross, Owen A. ; Caulfield, Thomas R. ; Springer, Wolfdieter ; Fiesel, Fabienne C.</creator><creatorcontrib>Bustillos, Bernardo A. ; Cocker, Liam T. ; Coban, Mathew A. ; Weber, Caleb A. ; Bredenberg, Jenny M. ; Boneski, Paige K. ; Siuda, Joanna ; Slawek, Jaroslaw ; Puschmann, Andreas ; Narendra, Derek P. ; Graff-Radford, Neill R. ; Wszolek, Zbigniew K. ; Dickson, Dennis W. ; Ross, Owen A. ; Caulfield, Thomas R. ; Springer, Wolfdieter ; Fiesel, Fabienne C.</creatorcontrib><description>Mutations in the PINK1 and PRKN genes are the most frequent genetic cause of early-onset Parkinson disease. The pathogenic p.R275W substitution in PRKN is the most frequent substitution observed in patients, and thus far has been characterized mostly through overexpression models that suggest a possible gain of toxic misfunction. However, its effects under endogenous conditions are largely unknown. We used patient fibroblasts, isogenic neurons, and post-mortem human brain samples from carriers with and without PRKN p.R275W to assess functional impact. Immunoblot analysis and immunofluorescence were used to study mitophagy activation, and mitophagy execution was analyzed by flow cytometry of the reporter mitoKeima. The functional analysis was accompanied by structural investigation of PRKN p.R275W. We observed lower PRKN protein in fibroblasts with compound heterozygous p.R275W mutations. Isogenic neurons showed an allele-dose dependent decrease in PRKN protein. Lower PRKN protein levels were accompanied by diminished phosphorylated ubiquitin and decreased MFN2 modification. Mitochondrial degradation was also allele-dose dependently impaired. Consistently, PRKN protein levels were drastically reduced in human brain samples from p.R275W carriers. Finally, structural simulations showed significant changes in the closed form of PRKN p.R275W. Our data suggest that under endogenous conditions the p.R275W mutation results in a loss-of-function by destabilizing PRKN.</description><identifier>ISSN: 2073-4409</identifier><identifier>EISSN: 2073-4409</identifier><identifier>DOI: 10.3390/cells13181540</identifier><language>eng</language><ispartof>Cells (Basel, Switzerland), 2024-09, Vol.13 (18), p.1540</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c770-255c1a2a2775ae6182c83184592dd2b3520ed4f68d472cd04c183af9e30653ab3</cites><orcidid>0000-0002-3201-8198 ; 0009-0009-6704-2284 ; 0000-0002-0340-660X ; 0000-0001-5487-1053 ; 0000-0001-6396-6759 ; 0000-0002-8696-9108 ; 0000-0002-1919-9676 ; 0000-0002-1178-3149 ; 0000-0002-1055-3069</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,786,790,27957,27958</link.rule.ids></links><search><creatorcontrib>Bustillos, Bernardo A.</creatorcontrib><creatorcontrib>Cocker, Liam T.</creatorcontrib><creatorcontrib>Coban, Mathew A.</creatorcontrib><creatorcontrib>Weber, Caleb A.</creatorcontrib><creatorcontrib>Bredenberg, Jenny M.</creatorcontrib><creatorcontrib>Boneski, Paige K.</creatorcontrib><creatorcontrib>Siuda, Joanna</creatorcontrib><creatorcontrib>Slawek, Jaroslaw</creatorcontrib><creatorcontrib>Puschmann, Andreas</creatorcontrib><creatorcontrib>Narendra, Derek P.</creatorcontrib><creatorcontrib>Graff-Radford, Neill R.</creatorcontrib><creatorcontrib>Wszolek, Zbigniew K.</creatorcontrib><creatorcontrib>Dickson, Dennis W.</creatorcontrib><creatorcontrib>Ross, Owen A.</creatorcontrib><creatorcontrib>Caulfield, Thomas R.</creatorcontrib><creatorcontrib>Springer, Wolfdieter</creatorcontrib><creatorcontrib>Fiesel, Fabienne C.</creatorcontrib><title>Structural and Functional Characterization of the Most Frequent Pathogenic PRKN Substitution p.R275W</title><title>Cells (Basel, Switzerland)</title><description>Mutations in the PINK1 and PRKN genes are the most frequent genetic cause of early-onset Parkinson disease. The pathogenic p.R275W substitution in PRKN is the most frequent substitution observed in patients, and thus far has been characterized mostly through overexpression models that suggest a possible gain of toxic misfunction. However, its effects under endogenous conditions are largely unknown. We used patient fibroblasts, isogenic neurons, and post-mortem human brain samples from carriers with and without PRKN p.R275W to assess functional impact. Immunoblot analysis and immunofluorescence were used to study mitophagy activation, and mitophagy execution was analyzed by flow cytometry of the reporter mitoKeima. The functional analysis was accompanied by structural investigation of PRKN p.R275W. We observed lower PRKN protein in fibroblasts with compound heterozygous p.R275W mutations. Isogenic neurons showed an allele-dose dependent decrease in PRKN protein. Lower PRKN protein levels were accompanied by diminished phosphorylated ubiquitin and decreased MFN2 modification. Mitochondrial degradation was also allele-dose dependently impaired. Consistently, PRKN protein levels were drastically reduced in human brain samples from p.R275W carriers. Finally, structural simulations showed significant changes in the closed form of PRKN p.R275W. Our data suggest that under endogenous conditions the p.R275W mutation results in a loss-of-function by destabilizing PRKN.</description><issn>2073-4409</issn><issn>2073-4409</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2024</creationdate><recordtype>article</recordtype><recordid>eNpVUMtOwzAQtBBIVKVH7v6BFD9j54giAogCVVuJY7SxHRIUkmI7B_j6psAB5jIPaVbaQeiSkiXnGbkyrusC5VRTKcgJmjGieCIEyU7_6HO0COGNTNA0pUTOkN1GP5o4eugw9BYXY29iO_STzRvwYKLz7RccIzzUODYOPw4h4sK7j9H1Ea8hNsOr61uD15uHJ7wdqxDbOH439ssNU_LlAp3V0AW3-OU52hU3u_wuWT3f3ufXq8QoRRImpaHAgCklwaVUM6Onf4TMmLWs4pIRZ0WdaisUM5YIQzWHOnOcpJJDxeco-Tlr_BCCd3W59-07-M-SkvI4UvlvJH4AAiJazQ</recordid><startdate>20240913</startdate><enddate>20240913</enddate><creator>Bustillos, Bernardo A.</creator><creator>Cocker, Liam T.</creator><creator>Coban, Mathew A.</creator><creator>Weber, Caleb A.</creator><creator>Bredenberg, Jenny M.</creator><creator>Boneski, Paige K.</creator><creator>Siuda, Joanna</creator><creator>Slawek, Jaroslaw</creator><creator>Puschmann, Andreas</creator><creator>Narendra, Derek P.</creator><creator>Graff-Radford, Neill R.</creator><creator>Wszolek, Zbigniew K.</creator><creator>Dickson, Dennis W.</creator><creator>Ross, Owen A.</creator><creator>Caulfield, Thomas R.</creator><creator>Springer, Wolfdieter</creator><creator>Fiesel, Fabienne C.</creator><scope>AAYXX</scope><scope>CITATION</scope><orcidid>https://orcid.org/0000-0002-3201-8198</orcidid><orcidid>https://orcid.org/0009-0009-6704-2284</orcidid><orcidid>https://orcid.org/0000-0002-0340-660X</orcidid><orcidid>https://orcid.org/0000-0001-5487-1053</orcidid><orcidid>https://orcid.org/0000-0001-6396-6759</orcidid><orcidid>https://orcid.org/0000-0002-8696-9108</orcidid><orcidid>https://orcid.org/0000-0002-1919-9676</orcidid><orcidid>https://orcid.org/0000-0002-1178-3149</orcidid><orcidid>https://orcid.org/0000-0002-1055-3069</orcidid></search><sort><creationdate>20240913</creationdate><title>Structural and Functional Characterization of the Most Frequent Pathogenic PRKN Substitution p.R275W</title><author>Bustillos, Bernardo A. ; Cocker, Liam T. ; Coban, Mathew A. ; Weber, Caleb A. ; Bredenberg, Jenny M. ; Boneski, Paige K. ; Siuda, Joanna ; Slawek, Jaroslaw ; Puschmann, Andreas ; Narendra, Derek P. ; Graff-Radford, Neill R. ; Wszolek, Zbigniew K. ; Dickson, Dennis W. ; Ross, Owen A. ; Caulfield, Thomas R. ; Springer, Wolfdieter ; Fiesel, Fabienne C.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c770-255c1a2a2775ae6182c83184592dd2b3520ed4f68d472cd04c183af9e30653ab3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2024</creationdate><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Bustillos, Bernardo A.</creatorcontrib><creatorcontrib>Cocker, Liam T.</creatorcontrib><creatorcontrib>Coban, Mathew A.</creatorcontrib><creatorcontrib>Weber, Caleb A.</creatorcontrib><creatorcontrib>Bredenberg, Jenny M.</creatorcontrib><creatorcontrib>Boneski, Paige K.</creatorcontrib><creatorcontrib>Siuda, Joanna</creatorcontrib><creatorcontrib>Slawek, Jaroslaw</creatorcontrib><creatorcontrib>Puschmann, Andreas</creatorcontrib><creatorcontrib>Narendra, Derek P.</creatorcontrib><creatorcontrib>Graff-Radford, Neill R.</creatorcontrib><creatorcontrib>Wszolek, Zbigniew K.</creatorcontrib><creatorcontrib>Dickson, Dennis W.</creatorcontrib><creatorcontrib>Ross, Owen A.</creatorcontrib><creatorcontrib>Caulfield, Thomas R.</creatorcontrib><creatorcontrib>Springer, Wolfdieter</creatorcontrib><creatorcontrib>Fiesel, Fabienne C.</creatorcontrib><collection>CrossRef</collection><jtitle>Cells (Basel, Switzerland)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bustillos, Bernardo A.</au><au>Cocker, Liam T.</au><au>Coban, Mathew A.</au><au>Weber, Caleb A.</au><au>Bredenberg, Jenny M.</au><au>Boneski, Paige K.</au><au>Siuda, Joanna</au><au>Slawek, Jaroslaw</au><au>Puschmann, Andreas</au><au>Narendra, Derek P.</au><au>Graff-Radford, Neill R.</au><au>Wszolek, Zbigniew K.</au><au>Dickson, Dennis W.</au><au>Ross, Owen A.</au><au>Caulfield, Thomas R.</au><au>Springer, Wolfdieter</au><au>Fiesel, Fabienne C.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Structural and Functional Characterization of the Most Frequent Pathogenic PRKN Substitution p.R275W</atitle><jtitle>Cells (Basel, Switzerland)</jtitle><date>2024-09-13</date><risdate>2024</risdate><volume>13</volume><issue>18</issue><spage>1540</spage><pages>1540-</pages><issn>2073-4409</issn><eissn>2073-4409</eissn><abstract>Mutations in the PINK1 and PRKN genes are the most frequent genetic cause of early-onset Parkinson disease. The pathogenic p.R275W substitution in PRKN is the most frequent substitution observed in patients, and thus far has been characterized mostly through overexpression models that suggest a possible gain of toxic misfunction. However, its effects under endogenous conditions are largely unknown. We used patient fibroblasts, isogenic neurons, and post-mortem human brain samples from carriers with and without PRKN p.R275W to assess functional impact. Immunoblot analysis and immunofluorescence were used to study mitophagy activation, and mitophagy execution was analyzed by flow cytometry of the reporter mitoKeima. The functional analysis was accompanied by structural investigation of PRKN p.R275W. We observed lower PRKN protein in fibroblasts with compound heterozygous p.R275W mutations. Isogenic neurons showed an allele-dose dependent decrease in PRKN protein. Lower PRKN protein levels were accompanied by diminished phosphorylated ubiquitin and decreased MFN2 modification. Mitochondrial degradation was also allele-dose dependently impaired. Consistently, PRKN protein levels were drastically reduced in human brain samples from p.R275W carriers. Finally, structural simulations showed significant changes in the closed form of PRKN p.R275W. Our data suggest that under endogenous conditions the p.R275W mutation results in a loss-of-function by destabilizing PRKN.</abstract><doi>10.3390/cells13181540</doi><orcidid>https://orcid.org/0000-0002-3201-8198</orcidid><orcidid>https://orcid.org/0009-0009-6704-2284</orcidid><orcidid>https://orcid.org/0000-0002-0340-660X</orcidid><orcidid>https://orcid.org/0000-0001-5487-1053</orcidid><orcidid>https://orcid.org/0000-0001-6396-6759</orcidid><orcidid>https://orcid.org/0000-0002-8696-9108</orcidid><orcidid>https://orcid.org/0000-0002-1919-9676</orcidid><orcidid>https://orcid.org/0000-0002-1178-3149</orcidid><orcidid>https://orcid.org/0000-0002-1055-3069</orcidid><oa>free_for_read</oa></addata></record>
fulltext fulltext
identifier ISSN: 2073-4409
ispartof Cells (Basel, Switzerland), 2024-09, Vol.13 (18), p.1540
issn 2073-4409
2073-4409
language eng
recordid cdi_crossref_primary_10_3390_cells13181540
source PubMed (Medline); Publicly Available Content Database
title Structural and Functional Characterization of the Most Frequent Pathogenic PRKN Substitution p.R275W
url http://sfxeu10.hosted.exlibrisgroup.com/loughborough?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-09-22T20%3A35%3A17IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-crossref&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Structural%20and%20Functional%20Characterization%20of%20the%20Most%20Frequent%20Pathogenic%20PRKN%20Substitution%20p.R275W&rft.jtitle=Cells%20(Basel,%20Switzerland)&rft.au=Bustillos,%20Bernardo%20A.&rft.date=2024-09-13&rft.volume=13&rft.issue=18&rft.spage=1540&rft.pages=1540-&rft.issn=2073-4409&rft.eissn=2073-4409&rft_id=info:doi/10.3390/cells13181540&rft_dat=%3Ccrossref%3E10_3390_cells13181540%3C/crossref%3E%3Cgrp_id%3Ecdi_FETCH-LOGICAL-c770-255c1a2a2775ae6182c83184592dd2b3520ed4f68d472cd04c183af9e30653ab3%3C/grp_id%3E%3Coa%3E%3C/oa%3E%3Curl%3E%3C/url%3E&rft_id=info:oai/&rft_id=info:pmid/&rfr_iscdi=true