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Interaction of huntingtin with PRMTs and its subsequent arginine methylation affects HTT solubility, phase transition behavior and neuronal toxicity.

ABSTRACT: Huntington's disease (HD) is an incurable neurodegenerative disorder caused by a CAG expansion in the huntingtin gene (HTT). Post-translational modifications of huntingtin protein (HTT), such as phosphorylation, acetylation and ubiquitination, have been implicated in HD pathogenesis. Arginine methylation/dimethylation is an important modification with an emerging role in neurodegeneration; however, arginine methylation of HTT remains largely unexplored. Here we report nearly two dozen novel arginine methylation/dimethylation sites on the endogenous HTT from human and mouse brain and human cells suggested by mass spectrometry with data-dependent acquisition. Targeted quantitative mass spectrometry identified differential arginine methylation at specific sites in HD patient-derived striatal precursor cell lines compared to normal controls. We found that HTT can interact with several type I protein arginine methyltransferases (PRMTs) via its N-terminal domain. Using a combination of in vitro methylation and cell-based experiments, we identified PRMT4 (CARM1) and PRMT6 as major enzymes methylating HTT at specific arginines. Alterations of these methylation sites had a profound effect on biochemical properties of HTT rendering it less soluble in cells and affected its liquid-liquid phase separation and phase transition patterns in vitro. We found that expanded HTT 1-586 fragment can form liquid-like assemblies, which converted into solid-like assemblies when the R200/205 methylation sites were altered. Methyl-null alterations increased HTT toxicity to neuronal cells, while overexpression of PRMT 4 and 6 was beneficial for neuronal survival. Thus, arginine methylation pathways that involve specific HTT-modifying PRMT enzymes and modulate HTT biochemical and toxic properties could provide targets for HD-modifying therapies.

SUBMITTER: Ratovitski T

PROVIDER: S-EPMC9122652 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Interaction of huntingtin with PRMTs and its subsequent arginine methylation affects HTT solubility, phase transition behavior and neuronal toxicity.

Ratovitski Tamara T Jiang Mali M O'Meally Robert N RN Rauniyar Priyanka P Chighladze Ekaterine E Faragó Anikó A Kamath Siddhi V SV Jin Jing J Shevelkin Alexey V AV Cole Robert N RN Ross Christopher A CA

Human molecular genetics 20220501 10

Huntington's disease (HD) is an incurable neurodegenerative disorder caused by a CAG expansion in the huntingtin gene (HTT). Post-translational modifications of huntingtin protein (HTT), such as phosphorylation, acetylation and ubiquitination, have been implicated in HD pathogenesis. Arginine methylation/dimethylation is an important modification with an emerging role in neurodegeneration; however, arginine methylation of HTT remains largely unexplored. Here we report nearly two dozen novel argi ...[more]

PMID: 34888656

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Project description:Current disease-modifying therapies for Huntington disease (HD) focus on lowering mutant HTT (huntingtin; mHTT) levels, and the immunosuppressant drug rapamycin is an intriguing therapeutic for aging and neurological disorders. Rapamycin interacts with FKBP1A/FKBP12 and FKBP5/FKBP51, inhibiting the MTORC1 complex and increasing cellular clearance mechanisms. Whether the levels of FKBP (FK506 binding protein) family members are altered in HD models and if these proteins are potential therapeutic targets for HD have not been investigated. Here, we found levels of FKBP5 are significantly reduced in HD R6/2 and zQ175 mouse models and human HD isogenic neural stem cells and medium spiny neurons derived from induced pluripotent stem cells. Moreover, FKBP5 interacts and colocalizes with HTT in the striatum and cortex of zQ175 mice and controls. Importantly, when we decreased FKBP5 levels or activity by genetic or pharmacological approaches, we observed reduced levels of mHTT in our isogenic human HD stem cell model. Decreasing FKBP5 levels by siRNA or pharmacological inhibition increased LC3-II levels and macroautophagic/autophagic flux, suggesting autophagic cellular clearance mechanisms are responsible for mHTT lowering. Unlike rapamycin, the effect of pharmacological inhibition with SAFit2, an inhibitor of FKBP5, is MTOR independent. Further, in vivo treatment for 2 weeks with SAFit2, results in reduced HTT levels in both HD R6/2 and zQ175 mouse models. Our studies establish FKBP5 as a protein involved in the pathogenesis of HD and identify FKBP5 as a potential therapeutic target for HD.Abbreviations : ACTB/β-actin: actin beta; AD: Alzheimer disease; BafA1: bafilomycin A1; BCA: bicinchoninic acid; BBB: blood brain barrier; BSA: bovine serum albumin; CoIP: co-immunoprecipitation; DMSO: dimethyl sulfoxide; DTT: dithiothreitol; FKBPs: FK506 binding proteins; HD: Huntington disease; HTT: huntingtin; iPSC: induced pluripotent stem cells; MAP1LC3/LC3:microtubule associated protein 1 light chain 3; MAPT/tau: microtubule associated protein tau; MES: 2-ethanesulfonic acid; MOPS: 3-(N-morphorlino)propanesulfonic acid); MSN: medium spiny neurons; mHTT: mutant huntingtin; MTOR: mechanistic target of rapamycin kinase; NSC: neural stem cells; ON: overnight; PD: Parkinson disease; PPIase: peptidyl-prolyl cis/trans-isomerases; polyQ: polyglutamine; PPP1R1B/DARPP-32: protein phosphatase 1 regulatory inhibitor subunit 1B; PTSD: post-traumatic stress disorder; RT: room temperature; SQSTM1/p62: sequestosome 1; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBST:Tris-buffered saline, 0.1% Tween 20; TUBA: tubulin; ULK1: unc-51 like autophagy activating kinase 1; VCL: vinculin; WT: littermate controls.

Project description:The precise degradation of dysfunctional mitochondria by mitophagy is essential for maintaining neuronal homeostasis. HTT (huntingtin) can interact with numerous other proteins and thereby perform multiple biological functions within the cell. In this study, we investigated the role of HTT during mitophagy and analyzed the impact of the expansion of its polyglutamine (polyQ) tract. HTT is involved in different mitophagy steps, promoting the physical proximity of different protein complexes during the initiation of mitophagy and recruiting mitophagy receptors essential for promoting the interaction between damaged mitochondria and the nascent autophagosome. The presence of the polyQ tract in mutant HTT affects the formation of these protein complexes and determines the negative consequences of mutant HTT on mitophagy, leading to the accumulation of damaged mitochondria and an increase in oxidative stress. These outcomes contribute to general mitochondrial dysfunction and neurodegeneration in Huntington disease.Abbreviations: AMPK: AMP-activated protein kinase; ATG13: autophagy related 13; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; CCCP: carbonyl cyanide 3-chlorophenyl hydrazone; DMEM: Dulbecco's modified eagle medium; EDTA: ethylene-diamine-tetra-acetic acid; EGFP: enhanced green fluorescent protein; EGTA: ethylene glycol bis(2-aminoethyl ether)tetraacetic acid; FUNDC1: FUN14 domain containing 1; HD: Huntington disease; HRP: horseradish peroxidase; HTT: huntingtin; LC3-II: lipidated form of MAP1LC3/LC3; mtDNA: mitochondrial deoxyribonucleic acid; MTDR: MitoTracker Deep Red; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1, autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; OCR: oxygen consumption rate; OPTN: optineurin; OXPHOS: oxidative phosphorylation; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PINK1: PTEN induced putative kinase 1; PLA: proximity ligation assay; PMSF: phenylmethylsulfonyl fluoride; polyQ: polyglutamine; PtdIns3K: phosphatidylinositol 3-kinase; ROS: reactive oxygen species; Rot: rotenone; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM: standard error of the mean; SQSTM1/p62: sequestosome 1; TMRM: tetramethylrhodamine methyl ester; UB: ubiquitin; ULK1: unc-51 like kinase 1.

Dataset Information

Interaction of huntingtin with PRMTs and its subsequent arginine methylation affects HTT solubility, phase transition behavior and neuronal toxicity.

Publications

Interaction of huntingtin with PRMTs and its subsequent arginine methylation affects HTT solubility, phase transition behavior and neuronal toxicity.

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