Order controls disordered droplets: structure-function relationships in C9orf72-derived poly(PR)

AJP Cell Physiology ◽

10.1152/ajpcell.00372.2021 ◽

2021 ◽

Author(s):

Kohsuke Kanekura ◽

Yuhei Hayamizu ◽

Masahiko Kuroda

Keyword(s):

Amino Acids ◽

Aromatic Amino Acids ◽

Polar Amino Acid ◽

Repeat Proteins ◽

Intron 1 ◽

Lateral Sclerosis ◽

Ran Translation ◽

Dipeptide Repeat ◽

Positively Charged

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have been thought as two distinct neurodegenerative diseases. However, recent genetic screening and careful investigations found the genetic and pathological overlap among these disorders. Hexanucleotide expansions in intron 1 of C9orf72 are a leading cause of familial ALS and familial FTD. These expansions facilitate the repeat-associated non-ATG initiated translation (RAN translation), producing five dipeptide repeat proteins (DRPs), including Arg-rich poly(PR: Pro-Arg) and poly-(GR: Gly-Arg) peptides. Arg is a positively charged, highly polar amino acid that facilitates interactions with anionic molecules such as nucleic acids and acidic amino acids via electrostatic forces and aromatic amino acids via cation-pi interaction, suggesting that Arg-rich DRPs underlie the pathophysiology of ALS via Arg-mediated molecular interactions. Arg-rich DRPs have also been reported to induce neurodegeneration in cellular and animal models via multiple mechanisms; however, it remains unclear why the Arg-rich DRPs exhibit such diverse toxic properties, because not all Arg-rich peptides are toxic. In this mini-review, we discuss the current understanding of the pathophysiology of Arg-rich C9orf72 DRPs and introduce recent findings on the role of Arg distribution as a determinant of the toxicity and its contribution to the pathogenesis of ALS.

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SFPQ regulates the accumulation of RNA foci and dipeptide repeat proteins from the expanded repeat mutation in C9orf72

Journal of Cell Science ◽

10.1242/jcs.256602 ◽

2021 ◽

Vol 134 (4) ◽

pp. jcs256602 ◽

Cited By ~ 1

Author(s):

Mirjana Malnar ◽

Boris Rogelj

Keyword(s):

Antisense Rna ◽

Rna Binding ◽

Dipeptide Repeat Proteins ◽

Rna Transcripts ◽

Repeat Proteins ◽

Rna Foci ◽

Hek Cells ◽

Lateral Sclerosis ◽

Dipeptide Repeat

ABSTRACTThe expanded GGGGCC repeat mutation in the C9orf72 gene is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansion is transcribed to sense and antisense RNA, which form RNA foci and bind cellular proteins. This mechanism of action is considered cytotoxic. Translation of the expanded RNA transcripts also leads to the accumulation of toxic dipeptide repeat proteins (DPRs). The RNA-binding protein splicing factor proline and glutamine rich (SFPQ), which is being increasingly associated with ALS and FTD pathology, binds to sense RNA foci. Here, we show that SFPQ plays an important role in the C9orf72 mutation. Overexpression of SFPQ resulted in higher numbers of both sense and antisense RNA foci and DPRs in transfected human embryonic kidney (HEK) cells. Conversely, reduced SPFQ levels resulted in lower numbers of RNA foci and DPRs in both transfected HEK cells and C9orf72 mutation-positive patient-derived fibroblasts and lymphoblasts. Therefore, we have revealed a role of SFPQ in regulating the C9orf72 mutation that has implications for understanding and developing novel therapeutic targets for ALS and FTD.This article has an associated First Person interview with the first author of the paper.

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Type I PRMT inhibition protects against C9ORF72 arginine-rich dipeptide repeat toxicity

10.1101/2020.05.20.106260 ◽

2020 ◽

Cited By ~ 2

Author(s):

Alan S. Premasiri ◽

Anna L. Gill ◽

Fernando G. Vieira

Keyword(s):

Repeat Expansion ◽

Type I ◽

Arginine Methyltransferase ◽

Repeat Proteins ◽

C9orf72 Gene ◽

Expansion Mutation ◽

Determining Factor ◽

Lateral Sclerosis ◽

Ran Translation ◽

Dipeptide Repeat

ABSTRACTThe most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a repeat expansion mutation in the C9orf72 gene. Repeat-associated non-AUG (RAN) translation of this expansion produces five species of dipeptide repeat proteins (DRPs). The arginine containing DRPs, polyGR and polyPR, are consistently reported to be the most toxic. Here, we uncover Type I protein arginine methyltransferase (PRMT) inhibitors as possible therapeutics for polyGR- and polyPR- related toxicity. Furthermore, we reveal data that suggest that asymmetric dimethylation (ADMe) of polyGR is a determining factor in its pathogenesis.

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Soluble and insoluble dipeptide repeat protein measurements in C9orf72-frontotemporal dementia brains show regional differential solubility and correlation of poly-GR with clinical severity

Acta Neuropathologica Communications ◽

10.1186/s40478-020-01036-y ◽

2020 ◽

Vol 8 (1) ◽

Author(s):

Annelies Quaegebeur ◽

Idoia Glaria ◽

Tammaryn Lashley ◽

Adrian M. Isaacs

Keyword(s):

Frontotemporal Dementia ◽

Potential Role ◽

Brain Regions ◽

Clinical Severity ◽

Repeat Proteins ◽

Differential Solubility ◽

Shed Light ◽

Lateral Sclerosis ◽

Dipeptide Repeat

Abstract A C9orf72 repeat expansion is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis. One of the suggested pathomechanisms is toxicity from dipeptide repeat proteins (DPRs), which are generated via unconventional translation of sense and antisense repeat transcripts with poly-GA, poly-GP and poly-GR being the most abundant dipeptide proteins. Animal and cellular studies highlight a neurotoxic role of poly-GR and poly-PR and to a lesser degree of poly-GA. Human post-mortem studies in contrast have been much less clear on a potential role of DPR toxicity but have largely focused on immunohistochemical methods to detect aggregated DPR inclusions. This study uses protein fractionation and sensitive immunoassays to quantify not only insoluble but also soluble poly-GA, poly-GP and poly-GR concentrations in brain homogenates of FTD patients with C9orf72 mutation across four brain regions. We show that soluble DPRs are less abundant in clinically affected areas (i.e. frontal and temporal cortices). In contrast, the cerebellum not only shows the largest DPR load but also the highest relative DPR solubility. Finally, poly-GR levels and poly-GP solubility correlate with clinical severity. These findings provide the first cross-comparison of soluble and insoluble forms of all sense DPRs and shed light on the distribution and role of soluble DPRs in the etiopathogenesis of human C9orf72-FTD.

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Hippocampal protein aggregation signatures fully distinguish pathogenic and wildtype UBQLN2 in amyotrophic lateral sclerosis

10.1101/2022.01.12.475792 ◽

2022 ◽

Author(s):

Kyrah M Thumbadoo ◽

Birger V Dieriks ◽

Helen C Murray ◽

Molly EV Swanson ◽

Ji Hun Yoo ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Protein Aggregation ◽

Gene Mutations ◽

Repeat Proteins ◽

Familial Als ◽

Fluorescent Immunohistochemistry ◽

Self Aggregation ◽

Lateral Sclerosis ◽

Dipeptide Repeat

Mutations in the UBQLN2 gene cause X-linked dominant amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD) characterised by ubiquilin 2 aggregates in neurons of the motor cortex, hippocampus, cerebellum, and spinal cord. However, ubiquilin 2 neuropathology is also seen in sporadic and familial ALS or FTD cases not caused by UBQLN2 mutations, particularly C9ORF72-linked cases. This makes the mechanistic role of ubiquilin 2 mutations and the value of ubiquilin 2 pathology for predicting genotype unclear. Here we examine a cohort of 31 genotypically diverse ALS cases with or without FTD, including four cases with UBQLN2 mutations (resulting in p.P497H, p.P506S, and two cases with p.T487I). Using double-, triple-, and six-label fluorescent immunohistochemistry, we mapped the co-localisation of ubiquilin 2 with phosphorylated TDP-43 (pTDP-43), dipeptide repeat aggregates, and p62, in the hippocampus of controls (n=5), or ALS with or without FTD in sporadic (n=19), unknown familial (n=3), SOD1-linked (n=1), C9ORF72-linked (n=4), and UBQLN2-linked (n=4) cases. We differentiate between i) ubiquilin 2 aggregation together with, or driven by, pTDP-43 or dipeptide repeat proteins, and ii) ubiquilin 2 self-aggregation driven by UBQLN2 gene mutations. Together we describe a hippocampal protein aggregation signature that fully distinguishes mutant from wildtype ubiquilin 2 in ALS with or without FTD, whereby mutant ubiquilin 2 is more prone than wildtype to aggregate independently of driving factors. This neuropathological signature can be used to assess the pathogenicity of UBQLN2 gene mutations and to understand the mechanisms of UBQLN2-linked disease.

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The nuclear ubiquitin ligase adaptor SPOP is a conserved regulator of C9orf72 dipeptide toxicity

10.1101/2021.03.09.434618 ◽

2021 ◽

Author(s):

Carley Snoznik ◽

Valentina Medvedeva ◽

Jelena Mojsilovic-Petrovic ◽

Paige Rudich ◽

James Oosten ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Nuclear Localization ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Repeat Expansion ◽

Dipeptide Repeat Proteins ◽

C Elegans ◽

Repeat Proteins ◽

Lateral Sclerosis ◽

Dipeptide Repeat

AbstractA hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Unconventional translation of the C9orf72 repeat produces dipeptide repeat proteins (DPRs). Previously, we showed that the DPRs (PR)50 and (GR)50 are highly toxic when expressed in C. elegans and this toxicity depends on nuclear localization of the DPR. In an unbiased genome-wide RNAi screen for suppressors of (PR)50 toxicity, we identified 12 genes that consistently suppressed either the developmental arrest and/or paralysis phenotype evoked by (PR)50 expression. All of these genes have vertebrate homologs and 7/12 contain predicted nuclear localization signals. One of these genes was spop-1, the C. elegans homolog of SPOP, a nuclear localized E3 ubiquitin ligase adaptor only found in metazoans. SPOP is also required for (GR)50 toxicity and functions in a genetic pathway that includes cul-3, which is the canonical E3 ligase partner for SPOP. Genetic or pharmacological inhibition of SPOP in mammalian primary spinal cord motor neurons suppressed DPR toxicity without affecting DPR expression levels. Finally, we find that genetic inhibition of bet-1, the C. elegans homolog of the known SPOP ubiquitination targets BRD2/3/4, suppresses the protective effect of SPOP mutations. Together, these data suggest a model in which SPOP promotes the DPR-dependent ubiquitination and degradation of BRD proteins. We speculate the pharmacological manipulation of this pathway, which is currently underway for multiple cancer subtypes, could also represent a novel entry point for therapeutic intervention to treat C9 FTD/ALS.Significance statementThe G4C2 repeat expansion in the C9orf72 gene is a major cause of Fronto-Temporal Dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS). Unusual translation of the repeat sequence produces two highly toxic dipeptide repeat proteins, PRX and GRX, which accumulate in the brain tissue of individuals with these diseases. Here, we show that PR and GR toxicity in both C. elegans and mammalian neurons depends on the E3 ubiquitin ligase adaptor SPOP. SPOP acts through the bromodomain protein BET-1 to mediate dipeptide toxicity. SPOP inhibitors, which are currently being developed to treat SPOP-dependent renal cancer, also protect neurons against DPR toxicity. Our findings identify a highly conserved and ‘druggable’ pathway that may represent a new strategy for treating these currently incurable diseases.

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Chain Elongation of Aromatic Amino Acids: the Role of 2-Benzylmalic Acid in the Biosynthesis of a C6C4 Amino Acid and a C6C3 Mustard Oil Glucoside

Canadian Journal of Biochemistry ◽

10.1139/o74-129 ◽

1974 ◽

Vol 52 (10) ◽

pp. 916-921 ◽

Cited By ~ 8

Author(s):

D. Dörnemann ◽

W. Löffelhardt ◽

H. Kindl

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Aromatic Amino Acids ◽

Chain Elongation ◽

Mustard Oil ◽

Barbarea Vulgaris ◽

Nasturtium Officinale ◽

A Chain ◽

Phenylbutyric Acid

A chemical synthesis of specifically 14C-labelled 2-benzylmalic acid, hitherto unknown, was developed. 4-Phenylacetoacetate obtained by condensation of phenylacetyl chloride-1-14C with ethyl acetoacetate yielded 2-benzylmalic acid-2-14C after cyanohydrin reaction and hydrolysis.2-Benzylmalic acid-2-14C, administered to shoots of Nasturtium officinale and Barbarea vulgaris, was shown to be an efficient precursor of the aglucone moiety of the mustard oil glucoside gluconasturtiin. The incorporation of radioactivity agreed well with the values reported for incorporation of 3-benzylmalic acid, but was considerably higher than that obtained after application of L-phenylalanine-U-14C. A conversion of 2-benzylmalic acid into 3-benzylmalic acid and 2-amino-4-phenylbutyric acid could also be demonstrated. These findings provide the final evidence for a chain-lengthening mechanism leading to homologous amino acids as proposed by Underbill and Wetter in 1966.

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