Unbiased label-free quantitative proteomics of Amyotrophic lateral sclerosis (ALS) mutations in CCNF reveals activation of the apoptosis pathway
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture, Neuronal Stem Cell
DISEASE(S): Frontotemporal Dementia,Amyotrophic Lateral Sclerosis
SUBMITTER: 
Albert Lee
LAB HEAD: Albert Lee
PROVIDER: PXD021793 | Pride | 2021-03-31
REPOSITORIES: Pride
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Project description:Approximately 10% of Amyotrophic lateral sclerosis (ALS) cases have a positive family history (familial ALS) and appear clinically indistinguishable from sporadic cases. ALS and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics; however, their biological mechanisms remain poorly understood. We have previously identified CCNF missense mutations in cohorts of familial and sporadic ALS and FTD cases [ref]. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase (SCFCyclin F) complex that is responsible for ubiquitinating proteins for degradation by the Ubiquitin-Proteasome System (UPS). The SCFCyclin F complex is essential for maintaining cellular homeostasis in cells; but mutations appear to lead to aberrant motor neuron development and neuron degeneration. We revealed elevated Lys48-specific ubiquitination of proteins in neuronal cells expressing mutant CCNFS621G compared to the CCNFWT control, which is consistent with increased Lys48-specific E3 ligase activity of cyclin FS621G (>1.3-fold). Different subsets of immunoprecipitated Lys48-ubiquitinated proteins were identified between CCNFWT and CCNFS621G cells indicating that transfected cyclin F contribute to the protein ubiquitination profile. These findings provide mechanistic insights into the effects of CCNF gene mutations on the function of the SCFcyclin F complex on neuronal proteostasis.
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Project description:Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by progressive weakness, paralysis and cachexia often resulting in patient death within 3-5 years of diagnosis. Recently, we identified mutations to the CCNF gene, which encodes the Cyclin F protein, in cohorts of patients with familial and sporadic ALS (1). Cyclin F is a part of a Skp1-Cul-F-box (SCF) E3 ubiquitin-protein ligase complex and is responsible for ubiquitinating proteins for the degradation by the proteasome. In this study, we investigated the phosphorylation status of Cyclin F and the effect of the serine mutation at site 621 to glycine on E3 ligase Lys48-specific ubiquitylation activity. We identified seven phosphorylation sites on Cyclin F, of which five were unique including Ser621. These phosphorylation sites were mostly identified within the PEST sequence of Cyclin F at the C-terminus. Additionally, we determined that Casein Kinase II (CK2) was responsible for phosphorylating Ser621 and controls the E3 ligase activity of the SCF(Cyclin F) complex. Thus, the glycine mutation at this site on Cyclin F prevents phosphorylation by CK2 and confers elevated Lys48-ubiquitylation activity, a hallmark of ALS pathogenesis. These findings highlight the convergence of post-translational modifications (ubiquitylation and phosphorylation) to sustain cellular homeostasis, and aberrations such as a single missense mutation can affect downstream cellular processes and lead to aberrant motor neuron development, neuron degeneration and ultimately ALS.
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