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Calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress
Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by loss-of-function mutations in the E3 ubiquitin ligase adaptor gigaxonin, which is encoded by the KLHL16 gene. Gigaxonin regulates the degradation of multiple intermediate filament (IF) proteins, including neurofilaments...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9664965/ https://www.ncbi.nlm.nih.gov/pubmed/36393840 http://dx.doi.org/10.3389/fcell.2022.1008542 |
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author | Phillips, Cassandra L. Fu, Dong Herring, Laura E. Armao, Diane Snider, Natasha T. |
author_facet | Phillips, Cassandra L. Fu, Dong Herring, Laura E. Armao, Diane Snider, Natasha T. |
author_sort | Phillips, Cassandra L. |
collection | PubMed |
description | Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by loss-of-function mutations in the E3 ubiquitin ligase adaptor gigaxonin, which is encoded by the KLHL16 gene. Gigaxonin regulates the degradation of multiple intermediate filament (IF) proteins, including neurofilaments, GFAP, and vimentin, which aggregate in GAN patient cells. Understanding how IFs and their aggregates are processed under stress can reveal new GAN disease mechanisms and potential targets for therapy. Here we tested the hypothesis that hypotonic stress-induced vimentin proteolysis is impaired in GAN. In both GAN and control fibroblasts exposed to hypotonic stress, we observed time-dependent vimentin cleavage that resulted in two prominent ∼40–45 kDa fragments. However, vimentin proteolysis occurred more rapidly and extensively in GAN cells compared to unaffected controls as both fragments were generated earlier and at 4-6-fold higher levels. To test enzymatic involvement, we determined the expression levels and localization of the calcium-sensitive calpain proteases-1 and -2 and their endogenous inhibitor calpastatin. While the latter was not affected, the expression of both calpains was 2-fold higher in GAN cells compared to control cells. Moreover, pharmacologic inhibition of calpains with MDL-28170 or MG-132 attenuated vimentin cleavage. Imaging analysis revealed striking colocalization between large perinuclear vimentin aggregates and calpain-2 in GAN fibroblasts. This colocalization was dramatically altered by hypotonic stress, where selective breakdown of filaments over aggregates occurred rapidly in GAN cells and coincided with calpain-2 cytoplasmic redistribution. Finally, mass spectrometry-based proteomics revealed that phosphorylation at Ser-412, located at the junction between the central “rod” domain and C-terminal “tail” domain on vimentin, is involved in this stress response. Over-expression studies using phospho-deficient and phospho-mimic mutants revealed that Ser-412 is important for filament organization, solubility dynamics, and vimentin cleavage upon hypotonic stress exposure. Collectively, our work reveals that osmotic stress induces calpain- and proteasome-mediated vimentin degradation and IF network breakdown. These effects are significantly augmented in the presence of disease-causing KLHL16 mutations that alter intermediate filament organization. While the specific roles of calpain-generated vimentin IF fragments in GAN cells remain to be defined, this proteolytic pathway is translationally-relevant to GAN because maintaining osmotic homeostasis is critical for nervous system function. |
format | Online Article Text |
id | pubmed-9664965 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-96649652022-11-15 Calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress Phillips, Cassandra L. Fu, Dong Herring, Laura E. Armao, Diane Snider, Natasha T. Front Cell Dev Biol Cell and Developmental Biology Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by loss-of-function mutations in the E3 ubiquitin ligase adaptor gigaxonin, which is encoded by the KLHL16 gene. Gigaxonin regulates the degradation of multiple intermediate filament (IF) proteins, including neurofilaments, GFAP, and vimentin, which aggregate in GAN patient cells. Understanding how IFs and their aggregates are processed under stress can reveal new GAN disease mechanisms and potential targets for therapy. Here we tested the hypothesis that hypotonic stress-induced vimentin proteolysis is impaired in GAN. In both GAN and control fibroblasts exposed to hypotonic stress, we observed time-dependent vimentin cleavage that resulted in two prominent ∼40–45 kDa fragments. However, vimentin proteolysis occurred more rapidly and extensively in GAN cells compared to unaffected controls as both fragments were generated earlier and at 4-6-fold higher levels. To test enzymatic involvement, we determined the expression levels and localization of the calcium-sensitive calpain proteases-1 and -2 and their endogenous inhibitor calpastatin. While the latter was not affected, the expression of both calpains was 2-fold higher in GAN cells compared to control cells. Moreover, pharmacologic inhibition of calpains with MDL-28170 or MG-132 attenuated vimentin cleavage. Imaging analysis revealed striking colocalization between large perinuclear vimentin aggregates and calpain-2 in GAN fibroblasts. This colocalization was dramatically altered by hypotonic stress, where selective breakdown of filaments over aggregates occurred rapidly in GAN cells and coincided with calpain-2 cytoplasmic redistribution. Finally, mass spectrometry-based proteomics revealed that phosphorylation at Ser-412, located at the junction between the central “rod” domain and C-terminal “tail” domain on vimentin, is involved in this stress response. Over-expression studies using phospho-deficient and phospho-mimic mutants revealed that Ser-412 is important for filament organization, solubility dynamics, and vimentin cleavage upon hypotonic stress exposure. Collectively, our work reveals that osmotic stress induces calpain- and proteasome-mediated vimentin degradation and IF network breakdown. These effects are significantly augmented in the presence of disease-causing KLHL16 mutations that alter intermediate filament organization. While the specific roles of calpain-generated vimentin IF fragments in GAN cells remain to be defined, this proteolytic pathway is translationally-relevant to GAN because maintaining osmotic homeostasis is critical for nervous system function. Frontiers Media S.A. 2022-11-01 /pmc/articles/PMC9664965/ /pubmed/36393840 http://dx.doi.org/10.3389/fcell.2022.1008542 Text en Copyright © 2022 Phillips, Fu, Herring, Armao and Snider. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Cell and Developmental Biology Phillips, Cassandra L. Fu, Dong Herring, Laura E. Armao, Diane Snider, Natasha T. Calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress |
title | Calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress |
title_full | Calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress |
title_fullStr | Calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress |
title_full_unstemmed | Calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress |
title_short | Calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress |
title_sort | calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress |
topic | Cell and Developmental Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9664965/ https://www.ncbi.nlm.nih.gov/pubmed/36393840 http://dx.doi.org/10.3389/fcell.2022.1008542 |
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