Cargando…

Development of a multiplexed targeted mass spectrometry assay for LRRK2-phosphorylated Rabs and Ser910/Ser935 biomarker sites

Mutations that increase the protein kinase activity of LRRK2 are one of the most common causes of familial Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif, impacting interaction with effectors. We describe and validate a new, multiplexed targeted m...

Descripción completa

Detalles Bibliográficos
Autores principales: Nirujogi, Raja S., Tonelli, Francesca, Taylor, Matthew, Lis, Pawel, Zimprich, Alexander, Sammler, Esther, Alessi, Dario R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Portland Press Ltd. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7833208/
https://www.ncbi.nlm.nih.gov/pubmed/33367571
http://dx.doi.org/10.1042/BCJ20200930
_version_ 1783642013303832576
author Nirujogi, Raja S.
Tonelli, Francesca
Taylor, Matthew
Lis, Pawel
Zimprich, Alexander
Sammler, Esther
Alessi, Dario R.
author_facet Nirujogi, Raja S.
Tonelli, Francesca
Taylor, Matthew
Lis, Pawel
Zimprich, Alexander
Sammler, Esther
Alessi, Dario R.
author_sort Nirujogi, Raja S.
collection PubMed
description Mutations that increase the protein kinase activity of LRRK2 are one of the most common causes of familial Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif, impacting interaction with effectors. We describe and validate a new, multiplexed targeted mass spectrometry assay to quantify endogenous levels of LRRK2-phosphorylated Rab substrates (Rab1, Rab3, Rab8, Rab10, Rab35 and Rab43) as well as total levels of Rabs, LRRK2 and LRRK2-phosphorylated at the Ser910 and Ser935 biomarker sites. Exploiting this assay, we quantify for the first time the relative levels of each of the pRab proteins in different cells (mouse embryonic fibroblasts, human neutrophils) and mouse tissues (brain, kidney, lung and spleen). We define how these components are impacted by Parkinson's pathogenic mutations (LRRK2[R1441C] and VPS35[D620N]) and LRRK2 inhibitors. We find that the VPS35[D620N], but not LRRK2[R1441C] mutation, enhances Rab1 phosphorylation in a manner blocked by administration of an LRRK2 inhibitor, providing the first evidence that endogenous Rab1 is a physiological substrate for LRRK2. We exploit this assay to demonstrate that in Parkinson's patients with VPS35[D620N] mutations, phosphorylation of multiple Rab proteins (Rab1, Rab3, Rab8, Rab10 and Rab43) is elevated. We highlight the benefits of this assay over immunoblotting approaches currently deployed to assess LRRK2 Rab signalling pathway.
format Online
Article
Text
id pubmed-7833208
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Portland Press Ltd.
record_format MEDLINE/PubMed
spelling pubmed-78332082021-02-02 Development of a multiplexed targeted mass spectrometry assay for LRRK2-phosphorylated Rabs and Ser910/Ser935 biomarker sites Nirujogi, Raja S. Tonelli, Francesca Taylor, Matthew Lis, Pawel Zimprich, Alexander Sammler, Esther Alessi, Dario R. Biochem J Signaling Mutations that increase the protein kinase activity of LRRK2 are one of the most common causes of familial Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif, impacting interaction with effectors. We describe and validate a new, multiplexed targeted mass spectrometry assay to quantify endogenous levels of LRRK2-phosphorylated Rab substrates (Rab1, Rab3, Rab8, Rab10, Rab35 and Rab43) as well as total levels of Rabs, LRRK2 and LRRK2-phosphorylated at the Ser910 and Ser935 biomarker sites. Exploiting this assay, we quantify for the first time the relative levels of each of the pRab proteins in different cells (mouse embryonic fibroblasts, human neutrophils) and mouse tissues (brain, kidney, lung and spleen). We define how these components are impacted by Parkinson's pathogenic mutations (LRRK2[R1441C] and VPS35[D620N]) and LRRK2 inhibitors. We find that the VPS35[D620N], but not LRRK2[R1441C] mutation, enhances Rab1 phosphorylation in a manner blocked by administration of an LRRK2 inhibitor, providing the first evidence that endogenous Rab1 is a physiological substrate for LRRK2. We exploit this assay to demonstrate that in Parkinson's patients with VPS35[D620N] mutations, phosphorylation of multiple Rab proteins (Rab1, Rab3, Rab8, Rab10 and Rab43) is elevated. We highlight the benefits of this assay over immunoblotting approaches currently deployed to assess LRRK2 Rab signalling pathway. Portland Press Ltd. 2021-01-29 2021-01-22 /pmc/articles/PMC7833208/ /pubmed/33367571 http://dx.doi.org/10.1042/BCJ20200930 Text en © 2021 The Author(s) https://creativecommons.org/licenses/by/4.0/ This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Signaling
Nirujogi, Raja S.
Tonelli, Francesca
Taylor, Matthew
Lis, Pawel
Zimprich, Alexander
Sammler, Esther
Alessi, Dario R.
Development of a multiplexed targeted mass spectrometry assay for LRRK2-phosphorylated Rabs and Ser910/Ser935 biomarker sites
title Development of a multiplexed targeted mass spectrometry assay for LRRK2-phosphorylated Rabs and Ser910/Ser935 biomarker sites
title_full Development of a multiplexed targeted mass spectrometry assay for LRRK2-phosphorylated Rabs and Ser910/Ser935 biomarker sites
title_fullStr Development of a multiplexed targeted mass spectrometry assay for LRRK2-phosphorylated Rabs and Ser910/Ser935 biomarker sites
title_full_unstemmed Development of a multiplexed targeted mass spectrometry assay for LRRK2-phosphorylated Rabs and Ser910/Ser935 biomarker sites
title_short Development of a multiplexed targeted mass spectrometry assay for LRRK2-phosphorylated Rabs and Ser910/Ser935 biomarker sites
title_sort development of a multiplexed targeted mass spectrometry assay for lrrk2-phosphorylated rabs and ser910/ser935 biomarker sites
topic Signaling
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7833208/
https://www.ncbi.nlm.nih.gov/pubmed/33367571
http://dx.doi.org/10.1042/BCJ20200930
work_keys_str_mv AT nirujogirajas developmentofamultiplexedtargetedmassspectrometryassayforlrrk2phosphorylatedrabsandser910ser935biomarkersites
AT tonellifrancesca developmentofamultiplexedtargetedmassspectrometryassayforlrrk2phosphorylatedrabsandser910ser935biomarkersites
AT taylormatthew developmentofamultiplexedtargetedmassspectrometryassayforlrrk2phosphorylatedrabsandser910ser935biomarkersites
AT lispawel developmentofamultiplexedtargetedmassspectrometryassayforlrrk2phosphorylatedrabsandser910ser935biomarkersites
AT zimprichalexander developmentofamultiplexedtargetedmassspectrometryassayforlrrk2phosphorylatedrabsandser910ser935biomarkersites
AT sammleresther developmentofamultiplexedtargetedmassspectrometryassayforlrrk2phosphorylatedrabsandser910ser935biomarkersites
AT alessidarior developmentofamultiplexedtargetedmassspectrometryassayforlrrk2phosphorylatedrabsandser910ser935biomarkersites