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.

Nascent mutant Huntingtin exon 1 chains do not stall on ribosomes during translation but aggregates do recruit machinery involved in ribosome quality control

“…The pFN21A-HaloTag 63 constructs were purchased from Promega. The P2A stall construct was prepared as described previously 64 including the Httex1 constructs [16]. with a ratio greater than one standard error from the mean were classified as HBRi whereas all cells with 103 a ratio smaller than one standard error from the mean were classified as PBRi.…”

Inclusion bodies formed by polyglutamine and poly(glycine-alanine) are enriched with distinct proteomes but converge in proteins that are risk factors for disease and involved in protein degradation

“…PolyGA appears less toxic than the others although has been reported in some models to confer toxicity [13-21]. We previously reported polyGA to be mildly toxic to cultured Neuro2a cells and to induce a distinct network of proteome changes that occur compared to the arg-rich DPRs [12]. We also noted a distinction to the other DPRs in forming large inclusions that morphologically are similar to the inclusions formed by polyQ.…”

“…A pEGFP-based construct expressing polyGA dipeptide repeat length of 101 dipeptides (polyGA 101 ) was generated as described previously [12]. This construct expresses a GFP fusion tag at N-terminus of the polyGA.…”

“…5 different DPRs are expressed, namely dipeptide polymers of glycine-alanine (polyGA), proline-arginine (polyPR), glycine-arginine (polyGR), proline-alanine (polyPA), and proline-glycine (PolyPG). Of these polyPR and polyGR are profoundly toxic when expressed in cell culture and animal models targeting mechanisms in ribosome biogenesis, translation, actin cytoskeleton among others [5-12]. PolyGA appears less toxic than the others although has been reported in some models to confer toxicity [13-21].…”

Immiscible inclusion bodies formed by polyglutamine and poly(glycine-alanine) are enriched with distinct proteomes but converge in proteins that are risk factors for disease and involved in protein degradation

“…5 different PDRs are expressed, namely dipeptide polymers of proline-arginine (polyPR), glycine-arginine (polyGR), proline-alanine (polyPA), proline-glycine (PolyPG) in addition to polyGA. Of these polyPR and polyGR are profoundly toxic when expressed in cell culture and animal models, with the toxicity targeting mechanisms in ribosome biogenesis, translation, and actin cytoskeleton among others [14][15][16][17][18][19][20][21]. PolyGA appears less toxic than the other PDRs although it has been reported to confer toxicity in some models [22][23][24][25][26][27][28][29][30].…”