Dipeptide repeat derived from C9orf72 hexanucleotide expansions forms amyloids or natively unfolded structures in vitro

Contrarily to the general believe, many biologically active proteins lack stable tertiary and/or secondary structure under physiological conditions in vitro. These intrinsically disordered proteins (IDPs) are highly abundant in nature and many of them are associated with various human diseases. The functional repertoire of IDPs complements the functions of ordered proteins. Since IDPs constitute a significant portion of any given proteome, they can be combined in an unfoldome; which is a portion of the proteome including all IDPs (also known as natively unfolded proteins, therefore, unfoldome), and describing their functions, structures, interactions, evolution, and so forth. Amino acid sequence and compositions of IDPs are very different from those of ordered proteins, making possible reliable identification of IDPs at the proteome level by various computational means. Furthermore, IDPs possess a number of unique structural properties and are characterized by a peculiar conformational behavior, including their high stability against low pH and high temperature and their structural indifference toward the unfolding by strong denaturants. These peculiarities were shown to be useful for elaboration of the experimental techniques for the large-scale identification of IDPs in various organisms. Some of the computational and experimental tools for the unfoldome discovery are discussed in this review.

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Phase separation and toxicity of C9orf72 poly(PR) depends on alternate distribution of arginine

The Journal of Cell Biology ◽

10.1083/jcb.202103160 ◽

2021 ◽

Vol 220 (11) ◽

Author(s):

Chen Chen ◽

Yoshiaki Yamanaka ◽

Koji Ueda ◽

Peiying Li ◽

Tamami Miyagi ◽

...

Keyword(s):

Phase Separation ◽

Charge Distribution ◽

Protein Interactions ◽

Protein Protein Interactions ◽

C9orf72 Gene ◽

And Diffusion ◽

Lateral Sclerosis ◽

Dipeptide Repeat ◽

Liquid Liquid Phase Separation

Arg (R)-rich dipeptide repeat proteins (DPRs; poly(PR): Pro-Arg and poly(GR): Gly-Arg), encoded by a hexanucleotide expansion in the C9ORF72 gene, induce neurodegeneration in amyotrophic lateral sclerosis (ALS). Although R-rich DPRs undergo liquid–liquid phase separation (LLPS), which affects multiple biological processes, mechanisms underlying LLPS of DPRs remain elusive. Here, using in silico, in vitro, and in cellulo methods, we determined that the distribution of charged Arg residues regulates the complex coacervation with anionic peptides and nucleic acids. Proteomic analyses revealed that alternate Arg distribution in poly(PR) facilitates entrapment of proteins with acidic motifs via LLPS. Transcription, translation, and diffusion of nucleolar nucleophosmin (NPM1) were impaired by poly(PR) with an alternate charge distribution but not by poly(PR) variants with a consecutive charge distribution. We propose that the pathogenicity of R-rich DPRs is mediated by disturbance of proteins through entrapment in the phase-separated droplets via sequence-controlled multivalent protein–protein interactions.

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Polymorphism in the procyclic acidic repetitive protein gene family of Trypanosoma brucei.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4055 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4055-4062 ◽

Cited By ~ 30

Author(s):

M R Mowatt ◽

C E Clayton

Keyword(s):

Molecular Weight ◽

Comparative Analysis ◽

Trypanosoma Brucei ◽

Untranslated Region ◽

Protein Gene ◽

Inverse Relation ◽

Repeat Domain ◽

Dipeptide Repeat ◽

Repetitive Protein

The expression of procyclic acidic repetitive protein (PARP) by Trypanosoma brucei is strongly induced during the transition of bloodstream form to cultured procyclic trypomastigotes in vitro. The membrane-associated protein is distinguished by a central domain consisting of tandemly repeated glutamate-proline dipeptides. The trypanosome genome contains eight PARP genes, at least four of which are expressed. A minimum of four distinct PARP mRNA species comprises two classes of PARP mRNA, based upon divergent 3' untranslated region sequences, and these mRNAs encode polypeptides that exhibited an inverse relation between molecular weight and isoelectric point. Comparative analysis of PARP gene structure indicated that these polypeptides differ by variation in size of the dipeptide repeat domain. Comparison of PARP genes and polypeptides of three independent T. brucei isolates suggested that PARP is not a homogeneous species but instead represents a family of polymorphic proteins.

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C9orf72 arginine-rich dipeptide repeat proteins disrupt karyopherin-mediated nuclear import

eLife ◽

10.7554/elife.51685 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 12

Author(s):

Lindsey R Hayes ◽

Lauren Duan ◽

Kelly Bowen ◽

Petr Kalab ◽

Jeffrey D Rothstein

Keyword(s):

Nuclear Import ◽

Genetic Modifier ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Dipeptide Repeat Proteins ◽

Hexanucleotide Repeat ◽

Repeat Proteins ◽

Importin Α ◽

Dipeptide Repeat

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases, including ALS caused by a C9orf72 hexanucleotide repeat expansion. However, the mechanism(s) remain unclear. Karyopherins, including importin β and its cargo adaptors, have been shown to co-precipitate with the C9orf72 arginine-containing dipeptide repeat proteins (R-DPRs), poly-glycine arginine (GR) and poly-proline arginine (PR), and are protective in genetic modifier screens. Here, we show that R-DPRs interact with importin β, disrupt its cargo loading, and inhibit nuclear import of importin β, importin α/β, and transportin cargoes in permeabilized mouse neurons and HeLa cells, in a manner that can be rescued by RNA. Although R-DPRs induce widespread protein aggregation in this in vitro system, transport disruption is not due to nucleocytoplasmic transport protein sequestration, nor blockade of the phenylalanine-glycine (FG)-rich nuclear pore complex. Our results support a model in which R-DPRs interfere with cargo loading on karyopherins.

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C9orf72 arginine-rich dipeptide repeat proteins disrupt importin β-mediated nuclear import

10.1101/787473 ◽

2019 ◽

Cited By ~ 1

Author(s):

Lindsey R. Hayes ◽

Lauren Duan ◽

Kelly Bowen ◽

Petr Kalab ◽

Jeffrey D. Rothstein

Keyword(s):

Nuclear Import ◽

Genetic Modifier ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Dipeptide Repeat Proteins ◽

Hexanucleotide Repeat ◽

Repeat Proteins ◽

Hexanucleotide Repeat Expansion ◽

Dipeptide Repeat

AbstractDisruption of nucleocytoplasmic transport (NCT), including mislocalization of the importin β cargo, TDP-43, is a hallmark of amyotrophic lateral sclerosis (ALS), including ALS caused by a hexanucleotide repeat expansion in C9orf72. However, the mechanism(s) remain unclear. Importin β and its cargo adaptors have been shown to co-precipitate with the C9orf72-arginine-containing dipeptide repeat proteins (R-DPRs), poly-glycine arginine (GR) and poly-proline arginine (PR), and are protective in genetic modifier screens. Here, we show that R-DPRs interact with importin β, disrupt its cargo loading, and inhibit nuclear import in permeabilized mouse neurons and HeLa cells, in a manner that can be rescued by RNA. Although R-DPRs induce widespread protein aggregation in this in vitro system, transport disruption is not due to NCT protein sequestration, nor blockade of the phenylalanine-glycine (FG)-rich nuclear pore complex. Our results support a model in which R-DPRs interfere with nuclear transport receptors in the vicinity of the nuclear envelope.

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C9-ALS/FTD-linked proline–arginine dipeptide repeat protein associates with paraspeckle components and increases paraspeckle formation

Cell Death and Disease ◽

10.1038/s41419-019-1983-5 ◽

2019 ◽

Vol 10 (10) ◽

Cited By ~ 4

Author(s):

Hiroaki Suzuki ◽

Yoshio Shibagaki ◽

Seisuke Hattori ◽

Masaaki Matsuoka

Keyword(s):

Dna Binding Protein ◽

Repeat Expansion ◽

Hexanucleotide Repeat ◽

Repeat Proteins ◽

Non Coding Rna ◽

Heterogeneous Nuclear Ribonucleoproteins ◽

Hexanucleotide Repeat Expansion ◽

Lateral Sclerosis ◽

Dipeptide Repeat

Abstract A GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce five dipeptide repeat proteins (DPRs). Although DPRs are thought to be neurotoxic, the molecular mechanism underlying the DPR-caused neurotoxicity has not been fully elucidated. The current study shows that poly-proline-arginine (poly-PR), the most toxic DPR in vitro, binds to and up-regulates nuclear paraspeckle assembly transcript 1 (NEAT1) that plays an essential role as a scaffold non-coding RNA during the paraspeckle formation. The CRISPR-assisted up-regulation of endogenous NEAT1 causes neurotoxicity. We also show that the poly-PR modulates the function of several paraspeckle-localizing heterogeneous nuclear ribonucleoproteins. Furthermore, dysregulated expression of TAR DNA-binding protein 43 (TDP-43) up-regulates NEAT1 expression and induces neurotoxicity. These results suggest that the increase in the paraspeckle formation may be involved in the poly-PR- and TDP-43-mediated neurotoxicity.

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Glycine-alanine dipeptide repeats spread rapidly in a repeat length- and age-dependent manner in the fly brain

Acta Neuropathologica Communications ◽

10.1186/s40478-019-0860-x ◽

2019 ◽

Vol 7 (1) ◽

Cited By ~ 4

Author(s):

Javier Morón-Oset ◽

Tessa Supèr ◽

Jacqueline Esser ◽

Adrian M. Isaacs ◽

Sebastian Grönke ◽

...

Keyword(s):

Dependent Manner ◽

Genetic Screens ◽

Hexanucleotide Repeat ◽

Repeat Expansions ◽

Alanine Dipeptide ◽

The Brain ◽

Lateral Sclerosis ◽

Dipeptide Repeat

AbstractHexanucleotide repeat expansions of variable size in C9orf72 are the most prevalent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Sense and antisense transcripts of the expansions are translated by repeat-associated non-AUG translation into five dipeptide repeat proteins (DPRs). Of these, the polyGR, polyPR and, to a lesser extent, polyGA DPRs are neurotoxic, with polyGA the most abundantly detected DPR in patient tissue. Trans-cellular transmission of protein aggregates has recently emerged as a major driver of toxicity in various neurodegenerative diseases. In vitro evidence suggests that the C9 DPRs can spread. However, whether this phenomenon occurs under more complex in vivo conditions remains unexplored. Here, we used the adult fly brain to investigate whether the C9 DPRs can spread in vivo upon expression in a subset of neurons. We found that only polyGA can progressively spread throughout the brain, which accumulates in the shape of aggregate-like puncta inside recipient cells. Interestingly, GA transmission occurred as early as 3 days after expression induction. By comparing the spread of 36, 100 and 200 polyGA repeats, we found that polyGA spread is enhanced upon expression of longer GA DPRs. Transmission of polyGA is greater in older flies, indicating that age-associated factors exacerbate the spread. These data highlight a unique propensity of polyGA to spread throughout the brain, which could contribute to the greater abundance of polyGA in patient tissue. In addition, we present a model of early GA transmission that is suitable for genetic screens to identify mechanisms of spread and its consequences in vivo.

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Polymorphism in the procyclic acidic repetitive protein gene family of Trypanosoma brucei

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4055-4062.1988 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4055-4062

Author(s):

M R Mowatt ◽

C E Clayton

Keyword(s):

Molecular Weight ◽

Comparative Analysis ◽

Trypanosoma Brucei ◽

Untranslated Region ◽

Protein Gene ◽

Inverse Relation ◽

Repeat Domain ◽

Dipeptide Repeat ◽

Repetitive Protein

The expression of procyclic acidic repetitive protein (PARP) by Trypanosoma brucei is strongly induced during the transition of bloodstream form to cultured procyclic trypomastigotes in vitro. The membrane-associated protein is distinguished by a central domain consisting of tandemly repeated glutamate-proline dipeptides. The trypanosome genome contains eight PARP genes, at least four of which are expressed. A minimum of four distinct PARP mRNA species comprises two classes of PARP mRNA, based upon divergent 3' untranslated region sequences, and these mRNAs encode polypeptides that exhibited an inverse relation between molecular weight and isoelectric point. Comparative analysis of PARP gene structure indicated that these polypeptides differ by variation in size of the dipeptide repeat domain. Comparison of PARP genes and polypeptides of three independent T. brucei isolates suggested that PARP is not a homogeneous species but instead represents a family of polymorphic proteins.

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C9orf72-derived poly-GA DPRs undergo endocytic uptake in iNPC-derived astrocytes and spread to motor neurons

10.1101/2021.10.11.463891 ◽

2021 ◽

Author(s):

Paolo M. Marchi ◽

Lara Marrone ◽

Laurent Brasseur ◽

Luc Bousset ◽

Christopher P. Webster ◽

...

Keyword(s):

Motor Neurons ◽

Disease Spread ◽

C9orf72 Gene ◽

Cell Propagation ◽

Β Sheet ◽

Lateral Sclerosis ◽

Dipeptide Repeat ◽

Axonal Swellings

Dipeptide repeat proteins (DPRs) are aggregation-prone polypeptides encoded by the pathogenic G4C2 repeat expansion in the C9orf72 gene, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). In this study, we focus on the role of poly-GA DPRs in disease spread. We demonstrate that recombinant poly-GA oligomers can directly convert into solid-like aggregates and form characteristic β-sheet fibrils in vitro. To dissect the process of cell-to-cell DPR transmission, we closely follow the fate of poly-GA DPRs in either their oligomeric or fibrillized form after administration in the cell culture medium. We observe that poly-GA DPRs are taken up via dynamin-dependent and -independent endocytosis, eventually converging at the lysosomal compartment and leading to axonal swellings in neurons. We then use a co-culture system to demonstrate astrocyte-to-motor neuron DPR propagation, showing that astrocytes may internalise and release aberrant peptides in disease pathogenesis. Overall, our results shed light on the mechanisms of poly-GA cellular uptake and cell-to-cell propagation, suggesting lysosomal impairment as a possible feature underlying the cellular pathogenicity of these DPR species.

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Cross-talk unfolded: MARCKS proteins

Biochemical Journal ◽

10.1042/bj3620001 ◽

2002 ◽

Vol 362 (1) ◽

pp. 1-12 ◽

Cited By ~ 140

Author(s):

Anna ARBUZOVA ◽

Arndt A. P. SCHMITZ ◽

Guy VERGÈRES

Keyword(s):

Actin Cytoskeleton ◽

Cross Talk ◽

Protein Modification ◽

Membrane Binding ◽

Point Of View ◽

Cellular Migration ◽

Effector Domain ◽

Kinase Substrate ◽

Natively Unfolded

The proteins of the MARCKS (myristoylated alanine-rich C kinase substrate) family were first identified as prominent substrates of protein kinase C (PKC). Since then, these proteins have been implicated in the regulation of brain development and postnatal survival, cellular migration and adhesion, as well as endo-, exo- and phago-cytosis, and neurosecretion. The effector domain of MARCKS proteins is phosphorylated by PKC, binds to calmodulin and contributes to membrane binding. This multitude of mutually exclusive interactions allows cross-talk between the signal transduction pathways involving PKC and calmodulin. This review focuses on recent, mostly biophysical and biochemical results renewing interest in this protein family. MARCKS membrane binding is now understood at the molecular level. From a structural point of view, there is a consensus emerging that MARCKS proteins are ‘natively unfolded'. Interestingly, domains similar to the effector domain have been discovered in other proteins. Furthermore, since the effector domain enhances the polymerization of actin in vitro, MARCKS proteins have been proposed to mediate regulation of the actin cytoskeleton. However, the recent observations that MARCKS might serve to sequester phosphatidylinositol 4,5-bisphosphate in the plasma membrane of unstimulated cells suggest an alternative model for the control of the actin cytoskeleton. While myristoylation is classically considered to be a co-translational, irreversible event, new reports on MARCKS proteins suggest a more dynamic picture of this protein modification. Finally, studies with mice lacking MARCKS proteins have investigated the functions of these proteins during embryonic development in the intact organism.

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