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Phosphorylation of huntingtin at residue T3 is decreased in Huntington's disease and modulates mutant huntingtin protein conformation.

ABSTRACT: Posttranslational modifications can have profound effects on the biological and biophysical properties of proteins associated with misfolding and aggregation. However, their detection and quantification in clinical samples and an understanding of the mechanisms underlying the pathological properties of misfolding- and aggregation-prone proteins remain a challenge for diagnostics and therapeutics development. We have applied an ultrasensitive immunoassay platform to develop and validate a quantitative assay for detecting a posttranslational modification (phosphorylation at residue T3) of a protein associated with polyglutamine repeat expansion, namely Huntingtin, and characterized its presence in a variety of preclinical and clinical samples. We find that T3 phosphorylation is greatly reduced in samples from Huntington's disease models and in Huntington's disease patients, and we provide evidence that bona-fide T3 phosphorylation alters Huntingtin exon 1 protein conformation and aggregation properties. These findings have significant implications for both mechanisms of disease pathogenesis and the development of therapeutics and diagnostics for Huntington's disease.

SUBMITTER: Cariulo C

PROVIDER: S-EPMC5740681 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Phosphorylation of huntingtin at residue T3 is decreased in Huntington's disease and modulates mutant huntingtin protein conformation.

Cariulo Cristina C Azzollini Lucia L Verani Margherita M Martufi Paola P Boggio Roberto R Chiki Anass A Deguire Sean M SM Cherubini Marta M Gines Silvia S Marsh J Lawrence JL Conforti Paola P Cattaneo Elena E Santimone Iolanda I Squitieri Ferdinando F Lashuel Hilal A HA Petricca Lara L Caricasole Andrea A

Proceedings of the National Academy of Sciences of the United States of America 20171121 50

Posttranslational modifications can have profound effects on the biological and biophysical properties of proteins associated with misfolding and aggregation. However, their detection and quantification in clinical samples and an understanding of the mechanisms underlying the pathological properties of misfolding- and aggregation-prone proteins remain a challenge for diagnostics and therapeutics development. We have applied an ultrasensitive immunoassay platform to develop and validate a quantit ...[more]

PMID: 29162692

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Project description:BackgroundQuantification of disease-associated proteins in the cerebrospinal fluid (CSF) has been critical for the study and treatment of several neurodegenerative disorders; however, mutant huntingtin protein (mHTT), the cause of Huntington's disease (HD), is at very low levels in CSF and, to our knowledge, has never been measured previously.MethodsWe developed an ultrasensitive single-molecule counting (SMC) mHTT immunoassay that was used to quantify mHTT levels in CSF samples from individuals bearing the HD mutation and from control individuals in 2 independent cohorts.ResultsThis SMC mHTT immunoassay demonstrated high specificity for mHTT, high sensitivity with a femtomolar detection threshold, and a broad dynamic range. Analysis of the CSF samples showed that mHTT was undetectable in CSF from all controls but quantifiable in nearly all mutation carriers. The mHTT concentration in CSF was approximately 3-fold higher in patients with manifest HD than in premanifest mutation carriers. Moreover, mHTT levels increased as the disease progressed and were associated with 5-year onset probability. The mHTT concentration independently predicted cognitive and motor dysfunction. Furthermore, the level of mHTT was associated with the concentrations of tau and neurofilament light chain in the CSF, suggesting a neuronal origin for the detected mHTT.ConclusionsWe have demonstrated that mHTT can be quantified in CSF from HD patients using the described SMC mHTT immunoassay. Moreover, the level of mHTT detected is associated with proximity to disease onset and diminished cognitive and motor function. The ability to quantify CSF mHTT will facilitate the study of HD, and mHTT quantification could potentially serve as a biomarker for the development and testing of experimental mHTT-lowering therapies for HD.Trial registrationNot applicable.FundingCHDI Foundation Inc.; Medical Research Council (MRC) UK; National Institutes for Health Research (NIHR); Rosetrees Trust; Swedish Research Council; and Knut and Alice Wallenberg Foundation.

Dataset Information

Phosphorylation of huntingtin at residue T3 is decreased in Huntington's disease and modulates mutant huntingtin protein conformation.

Publications

Phosphorylation of huntingtin at residue T3 is decreased in Huntington's disease and modulates mutant huntingtin protein conformation.

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