Pathogenic mutation in the ALS/FTD gene, CCNF, causes elevated Lys48-linked ubiquitylation and defective autophagy

Albert Lee, Stephanie L. Rayner, Serene S. L. Gwee, Alana De Luca, Hamideh Shahheydari, Vinod Sundaramoorthy, Audrey Ragagnin, Marco Morsch, Rowan Radford, Jasmin Galper, Sarah Freckleton, Bingyang Shi, Adam K. Walker, Emily K. Don, Nicholas J. Cole, Shu Yang, Kelly L. Williams, Justin J. Yerbury, Ian P. Blair, Julie D. Atkin & 2 others Mark P. Molloy, Roger S. Chung

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin–protein ligase (SCFcyclin F) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin–proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin FS621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin FWT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin FS621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin FS621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal–lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin FS621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.

LanguageEnglish
Pages335–354
Number of pages20
JournalCellular and Molecular Life Sciences
Volume75
Issue number2
Early online date2017
DOIs
Publication statusPublished - Jan 2018

Fingerprint

Cyclins
Ubiquitination
Autophagy
Mutation
Genes
Lysosome-Associated Membrane Glycoproteins
Chaperonins
Frontotemporal Dementia With Motor Neuron Disease
Frontotemporal Dementia
Proteins
Ubiquitin-Protein Ligases
Amyotrophic Lateral Sclerosis
Missense Mutation
Proteasome Endopeptidase Complex
Mutant Proteins
Ubiquitin
Heat-Shock Proteins
Lysosomes
Neurodegenerative Diseases
Proteomics

Keywords

  • Ubiquitylation
  • Phosphorylation
  • CCNF
  • Cyclin F
  • Amyotrophic lateral sclerosis
  • Frontotemporal dementia
  • Motor neuron disease

Cite this

@article{3d52817c834a404aba673de428204058,
title = "Pathogenic mutation in the ALS/FTD gene, CCNF, causes elevated Lys48-linked ubiquitylation and defective autophagy",
abstract = "Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin–protein ligase (SCFcyclin F) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin–proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin FS621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin FWT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin FS621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin FS621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal–lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin FS621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.",
keywords = "Ubiquitylation, Phosphorylation, CCNF, Cyclin F, Amyotrophic lateral sclerosis, Frontotemporal dementia, Motor neuron disease",
author = "Albert Lee and Rayner, {Stephanie L.} and Gwee, {Serene S. L.} and {De Luca}, Alana and Hamideh Shahheydari and Vinod Sundaramoorthy and Audrey Ragagnin and Marco Morsch and Rowan Radford and Jasmin Galper and Sarah Freckleton and Bingyang Shi and Walker, {Adam K.} and Don, {Emily K.} and Cole, {Nicholas J.} and Shu Yang and Williams, {Kelly L.} and Yerbury, {Justin J.} and Blair, {Ian P.} and Atkin, {Julie D.} and Molloy, {Mark P.} and Chung, {Roger S.}",
year = "2018",
month = "1",
doi = "10.1007/s00018-017-2632-8",
language = "English",
volume = "75",
pages = "335–354",
journal = "Cellular and Molecular Life Sciences",
issn = "1420-682X",
number = "2",

}

Pathogenic mutation in the ALS/FTD gene, CCNF, causes elevated Lys48-linked ubiquitylation and defective autophagy. / Lee, Albert; Rayner, Stephanie L.; Gwee, Serene S. L.; De Luca, Alana; Shahheydari, Hamideh; Sundaramoorthy, Vinod; Ragagnin, Audrey; Morsch, Marco; Radford, Rowan; Galper, Jasmin; Freckleton, Sarah; Shi, Bingyang; Walker, Adam K.; Don, Emily K.; Cole, Nicholas J.; Yang, Shu; Williams, Kelly L.; Yerbury, Justin J.; Blair, Ian P.; Atkin, Julie D.; Molloy, Mark P.; Chung, Roger S.

In: Cellular and Molecular Life Sciences, Vol. 75, No. 2, 01.2018, p. 335–354.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Pathogenic mutation in the ALS/FTD gene, CCNF, causes elevated Lys48-linked ubiquitylation and defective autophagy

AU - Lee, Albert

AU - Rayner, Stephanie L.

AU - Gwee, Serene S. L.

AU - De Luca, Alana

AU - Shahheydari, Hamideh

AU - Sundaramoorthy, Vinod

AU - Ragagnin, Audrey

AU - Morsch, Marco

AU - Radford, Rowan

AU - Galper, Jasmin

AU - Freckleton, Sarah

AU - Shi, Bingyang

AU - Walker, Adam K.

AU - Don, Emily K.

AU - Cole, Nicholas J.

AU - Yang, Shu

AU - Williams, Kelly L.

AU - Yerbury, Justin J.

AU - Blair, Ian P.

AU - Atkin, Julie D.

AU - Molloy, Mark P.

AU - Chung, Roger S.

PY - 2018/1

Y1 - 2018/1

N2 - Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin–protein ligase (SCFcyclin F) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin–proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin FS621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin FWT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin FS621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin FS621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal–lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin FS621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.

AB - Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin–protein ligase (SCFcyclin F) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin–proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin FS621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin FWT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin FS621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin FS621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal–lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin FS621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.

KW - Ubiquitylation

KW - Phosphorylation

KW - CCNF

KW - Cyclin F

KW - Amyotrophic lateral sclerosis

KW - Frontotemporal dementia

KW - Motor neuron disease

UR - http://www.scopus.com/inward/record.url?scp=85028760989&partnerID=8YFLogxK

UR - http://purl.org/au-research/grants/nhmrc/1107644

UR - http://purl.org/au-research/grants/nhmrc/1030513

UR - http://purl.org/au-research/grants/nhmrc/1095215

UR - http://purl.org/au-research/grants/nhmrc/1036835

U2 - 10.1007/s00018-017-2632-8

DO - 10.1007/s00018-017-2632-8

M3 - Article

VL - 75

SP - 335

EP - 354

JO - Cellular and Molecular Life Sciences

T2 - Cellular and Molecular Life Sciences

JF - Cellular and Molecular Life Sciences

SN - 1420-682X

IS - 2

ER -