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Arginine in C9ORF72 Dipolypeptides Mediates Promiscuous Proteome Binding and Multiple Modes of Toxicity.

ABSTRACT: C9ORF72-associated Motor Neuron Disease patients feature abnormal expression of 5 dipeptide repeat (DPR) polymers. Here we used quantitative proteomics in a mouse neuronal-like cell line (Neuro2a) to demonstrate that the Arg residues in the most toxic DPRS, PR and GR, leads to a promiscuous binding to the proteome compared with a relative sparse binding of the more inert AP and GA. Notable targets included ribosomal proteins, translation initiation factors and translation elongation factors. PR and GR comprising more than 10 repeats appeared to robustly stall on ribosomes during translation suggesting Arg-rich peptide domains can electrostatically jam the ribosome exit tunnel during synthesis. Poly-GR also recruited arginine methylases, induced hypomethylation of endogenous proteins, and induced a profound destabilization of the actin cytoskeleton. Our findings point to arginine in GR and PR polymers as multivalent toxins to translation as well as arginine methylation that may explain the dysfunction of biological processes including ribosome biogenesis, mRNA splicing and cytoskeleton assembly.

SUBMITTER: Radwan M

PROVIDER: S-EPMC7124463 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Arginine valency in C9ORF72 dipolypeptides mediates promiscuous proteome binding that transmits multiple modes of toxicity

Project description:C9ORF72-associated Motor Neuron Disease patients feature abnormal expression of 5 dipeptide repeat (DPR) polymers. Here we used quantitative proteomics in a mouse neuronal-like cell line (Neuro2a) to demonstrate that the valency of Arg in the most toxic DPRS, PR and GR, drives promiscuous binding to the proteome, compared to a relative sparse binding of the more inert AP and GA. Notable targets included ribosomal proteins, translation initiation factors and translation elongation factors. PR and GR comprising more than 10 repeats robustly stalled the ribosome suggesting high-valency Arg electrostatically jams the ribosome exit tunnel during synthesis. Poly-GR also bound to arginine methylases and induced hypomethylation of endogenous proteins, with a profound destabilization of the actin cytoskeleton. Our findings point to arginine in GR and PR polymers as multivalent toxins to translation as well as arginine methylation with concomitant downstream effects on widespread biological processes including ribosome biogenesis, mRNA splicing and cytoskeleton assembly.

2020-02-25 | PXD015180 | Pride

Arginine valency in C9ORF72 dipolypeptides mediates promiscuous proteome binding that transmits multiple modes of toxicity

Project description:Motor Neuron Disease patients with the C9ORF72 hexanucleotide expansion mutations feature abnormal expression of 5 different dipeptide repeat polymers (DPRs). Two of these, poly-GR and poly-PR, have proven highly toxic to cell and animal models. To investigate the mechanisms, we defined the interactomes of the DPRs in 10× and 101× repeat lengths as fusions to GFP in Neuro2a cells using quantitative proteomics. Relative to the more inert poly-GA and poly-PA peptides, poly-PR and poly-GR of both repeat lengths were promiscuous binders to the proteome and especially ribosomal proteins, translation initiation factors and translation elongation factors including ribosome recycling factor ABCE1, suggesting a role in ribosome stalling. The PR101 and GR101 DPRs robustly stalled the ribosome compared to the other DPRs and an unrelated mutant protein (Huntingtin) that causes neurodegeneration. Poly-GR also bound to arginine methylases and led to hypomethylation of endogenous proteins, with a profound destabilization of the actin cytoskeleton. Our findings point to arginine in the GR and PR polymers as multivalent toxins to translation as well as arginine methylation with concomitant downstream effects on widespread biological processes including ribosome biogenesis, mRNA splicing and cytoskeleton assembly.

2020-02-25 | PXD015177 | Pride

Type I PRMT Inhibition Protects Against C9ORF72 Arginine-Rich Dipeptide Repeat Toxicity.

Project description:Repeat expansion mutations in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-associated non-AUG translation of this expansion produces dipeptide repeat proteins (DRPs). The arginine containing DRPs, polyGR and polyPR, are consistently reported to be the most toxic. Here we demonstrated that small molecule inhibition of type I protein arginine methyltransferases (PRMT) protects against polyGR and polyPR toxicity. Furthermore, our findings suggest that asymmetric dimethylation of polyGR and polyPR by Type I PRMTs plays important roles in their cytotoxicity.

| S-EPMC7508178 | biostudies-literature

Nuclear localized C9orf72-associated arginine-containing dipeptides exhibit age-dependent toxicity in C. elegans.

Project description:A hexanucleotide repeat expansion mutation in the C9orf72 gene represents a prevalent genetic cause of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Non-canonical translation of this repeat gives rise to several distinct dipeptide protein species that could play pathological roles in disease. Here, we show in the model system Caenorhabditis elegans that expression of the arginine-containing dipeptides, but not alanine-containing dipeptides, produces toxic phenotypes in multiple cellular contexts, including motor neurons. Expression of either (PR)50 or (GR)50 during development caused a highly penetrant developmental arrest, while post-developmental expression caused age-onset paralysis. Both (PR)50- and (GR)50-green fluorescent protein tagged dipeptides were present in the nucleus and nuclear localization was necessary and sufficient for their toxicity. Using an inducible expression system, we discovered that age-onset phenotypes caused by (PR)50 required both continual (PR)50 expression and an aged cellular environment. The toxicity of (PR)50 was modified by genetic mutations that uncouple physiological aging from chronological aging. However, these same mutations failed to modify the toxicity of (GR)50, suggesting that (PR)50 and (GR)50 exert their toxicity through partially distinct mechanism(s). Changing the rate of physiological aging also mitigates toxicity in other C. elegans models of ALS, suggesting that the (PR)50 dipeptide might engage similar toxicity mechanisms as other ALS disease-causing proteins.

| S-EPMC5886095 | biostudies-literature

Poly(ADP-ribose) promotes toxicity of <i>C9ORF72</i> arginine-rich dipeptide repeat proteins.

Project description:Arginine-rich dipeptide repeat proteins (R-DPRs), abnormal translational products of a GGGGCC hexanucleotide repeat expansion in C9ORF72, play a critical role in C9ORF72-related amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the most common genetic form of the disorders (c9ALS/FTD). R-DPRs form liquid condensates in vitro, induce stress granule formation in cultured cells, aggregate, and sometimes coaggregate with TDP-43 in postmortem tissue from patients with c9ALS/FTD. However, how these processes are regulated is unclear. Here, we show that loss of poly(ADP-ribose) (PAR) suppresses neurodegeneration in c9ALS/FTD fly models and neurons differentiated from patient-derived induced pluripotent stem cells. Mechanistically, PAR induces R-DPR condensation and promotes R-DPR-induced stress granule formation and TDP-43 aggregation. Moreover, PAR associates with insoluble R-DPR and TDP-43 in postmortem tissue from patients. These findings identified PAR as a promoter of R-DPR toxicity and thus a potential target for treating c9ALS/FTD.

| S-EPMC10359073 | biostudies-literature

Reduced C9orf72 expression exacerbates polyGR toxicity in patient iPSC-derived motor neurons and a Type I protein arginine methyltransferase inhibitor reduces that toxicity

Project description:Introduction Intronic repeat expansions in the C9orf72 gene are the most frequent known single genetic causes of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These repeat expansions are believed to result in both loss-of-function and toxic gain-of-function. Gain-of-function results in the production of toxic arginine-rich dipeptide repeat proteins (DPRs), namely polyGR and polyPR. Small-molecule inhibition of Type I protein arginine methyltransferases (PRMTs) has been shown to protect against toxicity resulting from polyGR and polyPR challenge in NSC-34 cells and primary mouse-derived spinal neurons, but the effect in human motor neurons (MNs) has not yet been explored. Methods To study this, we generated a panel of C9orf72 homozygous and hemizygous knockout iPSCs to examine the contribution of C9orf72 loss-of-function toward disease pathogenesis. We differentiated these iPSCs into spinal motor neurons (sMNs). Results We found that reduced levels of C9orf72 exacerbate polyGR15 toxicity in a dose-dependent manner. Type I PRMT inhibition was able to partially rescue polyGR15 toxicity in both wild-type and C9orf72-expanded sMNs. Discussion This study explores the interplay of loss-of-function and gain-of-function toxicity in C9orf72 ALS. It also implicates type I PRMT inhibitors as a possible modulator of polyGR toxicity.

| S-EPMC10149854 | biostudies-literature

Improved ligand-protein binding affinity predictions using multiple binding modes.

Project description:Accurate ligand-protein binding affinity prediction, for a set of similar binders, is a major challenge in the lead optimization stage in drug development. In general, docking and scoring functions perform unsatisfactorily in this application. Docking calculations, followed by molecular dynamics simulations and free energy calculations can be applied to improve the predictions. However, for targets with large, flexible binding sites, with no experimentally determined binding modes for a set of ligands, insufficient sampling can decrease the accuracy of the free energy calculations. Cytochrome P450s, a protein family of major importance for drug metabolism, is an example of a challenging target for binding affinity predictions. As a result, the choice of starting structure from the docking solutions becomes crucial. In this study, an iterative scheme is introduced that includes multiple independent molecular dynamics simulations to obtain weighted ensemble averages to be used in the linear interaction energy method. The proposed scheme makes the initial pose selection less crucial for further simulation, as it automatically calculates the relative weights of the various poses. It also properly takes into account the possibility that multiple binding modes contribute similarly to the overall affinity, or of similar compounds occupying very different poses. The method was applied to a set of 12 compounds binding to cytochrome P450 2C9 and it displayed a root mean-square error of 2.9 kJ/mol.

| S-EPMC2877349 | biostudies-literature

Multiple drug binding modes in Mycobacterium tuberculosis CYP51B1.

Project description:The Mycobacterium tuberculosis (Mtb) genome encodes 20 different cytochrome P450 enzymes (CYPs), many of which serve essential biosynthetic roles. CYP51B1, the Mtb version of eukaryotic sterol demethylase, remains a potential therapeutic target. The binding of three drug fragments containing nitrogen heterocycles to CYP51B1 is studied here by continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) techniques to determine how each drug fragment binds to the heme active-site. All three drug fragments form a mixture of complexes, some of which retain the axial water ligand from the resting state. Hyperfine sublevel correlation spectroscopy (HYSCORE) and electron-nuclear double resonance spectroscopy (ENDOR) observe protons of the axial water and on the drug fragments that reveal drug binding modes. Binding in CYP51B1 is complicated by the presence of multiple binding modes that coexist in the same solution. These results aid our understanding of CYP-inhibitor interactions and will help guide future inhibitor design.

| S-EPMC7092363 | biostudies-literature

Conformational heterogeneity suggests multiple substrate binding modes in CYP106A2.

Project description:CYP106A2 (cytochrome P450meg) is a bacterial enzyme originally isolated from B. megaterium, and has been shown to hydroxylate a wide variety of substrates, including steroids. The regio- and stereochemistry of CYP106A2 hydroxylation has been shown to be dependent on a variety of factors, and hydroxylation often occurs at more than one site and/or with lack of stereospecificity for some substrates. Comprehensive backbone 15N, 1H and 13C resonance assignments based on multidimensional nuclear magnetic resonance (NMR) experiments performed with uniform and selective isotopically labeled CYP106A2 samples are reported herein, and broadening and splitting of resonances assigned to regions of the enzyme shown to be affected by substrate binding in other P450 enzymes indicate that substrate binding does not reduce structural heterogeneity as has been observed previously in P450 enzymes CYP101A1 and MycG. Paramagnetic relaxation enhancement (PRE) due to proximity between substrate protons and the heme iron were measured for three different substrates, and the relatively uniform nature of the PREs support the proposal that multiple substrate binding modes are occupied at saturating substrate concentrations.

| S-EPMC9992128 | biostudies-literature

C9orf72-derived arginine-rich poly-dipeptides impede phase modifiers.

Project description:Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-β2 (Kapβ2) at 1:1 ratio. The nuclear magnetic resonances of Kapβ2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapβ2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration.

| S-EPMC8421406 | biostudies-literature

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