Ultrasensitive quantitative measurement of huntingtin phosphorylation at residue S13

Cariulo, Cristina; Verani, Margherita; Martufi, Paola; Ingenito, Raffaele; Finotto, Marco; DeGuire, Sean M.; Lavery, Daniel; Toledo-Sherman, Leticia; Lee, Ramee; Doherty, Elizabeth M.; Vogt, Thomas; Dominguez, Celia; Lashuel, Hilal A.; Petricca, Lara; Caricasole, Andrea

doi:10.1016/j.bbrc.2019.09.097

Cited by 14 publications

(42 citation statements)

References 29 publications

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“…Therefore, we sought to examine whether TBK1 could phosphorylate longer N-terminal fragments and full-length HTT proteins. We co-expressed either the mutant HTTN548 (Q55) fragment or a full-length mutant HTT (Q48) with TBK1 or TBK1 KD in HEK293T cells and analyzed S13 phosphorylation by WB using an HTT pS13 antibody and using a new, sensitive Singulex assay, both of which we recently reported (Cristina Cariulo, 2019). We observed by WB that TBK1 phosphorylated both the HTTN548 Q55 fragment ( Fig.…”

Section: Resultsmentioning

confidence: 73%

“…1A-C). After an extensive in vitro validation using the top kinase hits from the screen, which included monitoring of the extent of phosphorylation using mass spectrometry as well as by the use of previously validated phospho-antibodies against T3 (pT3), S13 (pS13) and S16 (pS16) (Bustamante et al, 2015;Cristina Cariulo, 2019;Deguire et al, 2018), TBK1 was identified as the only kinase that selectively and robustly phosphorylated HTTex1 in vitro at S13 and S16 (Fig. 1C).…”

Section: Resultsmentioning

confidence: 99%

“…It was previously shown that mutant HTT from HD mouse model-derived cells shows less phosphorylation at S13/S16 than wild-type HTT (Atwal et al, 2011), and that pS13 HTT levels in a mouse striatal cell HD model as well as in HD knock-in mice are lower relative to wild type controls (Cristina Cariulo, 2019). To assess the role of phosphorylation in HTT pathology induction, we examined S13 phosphorylation levels in the brain in the zQ175 mouse model (Menalled et al, 2012).…”

Section: Consistent With Our Observation Inmentioning

confidence: 99%

“…psychiatric-like behavioral deficits, mutant HTT aggregation and selective neurodegeneration (Gu et al, 2009); and 3) the levels of phosphorylation at S13/S16 are reduced in HD models (Atwal et al, 2011;Cristina Cariulo, 2019). These observations suggest that targeting HTT PTMs at N17 represents a viable strategy for developing novel disease-modifying therapies for HD.…”

mentioning

confidence: 95%

“…TBK1 is known to regulate the innate immune response and belongs to the IκB kinase family that includes other kinases such as IKKβ, which was previously shown to phosphorylate HTT at these residues as well as at residue T3 (Bustamante et al, 2015), and was recently shown to modulate HTT S13 phosphorylation in HD mice (Ochaba et al, 2019). In this work, the specificity and efficiency of TBK1 for phosphorylating full-length and N-terminal fragments of HTT (HTTex1, HTTN548, HTT-fl) were validated in mammalian cells, primary neuronal culture and in vivo via overexpression of the wild-type and kinase-dead variant of TBK1, through a combination of techniques including a novel ultrasensitive immunoassay for pS13 HTT detection (Cristina Cariulo, 2019). Next, we assessed the effect of TBK1-mediated phosphorylation on HTT levels, subcellular localization, aggregation, and toxicity in cellular, neuronal and Caenorhabditis elegans (C. elegans) models of HD.…”

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confidence: 99%

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TBK1 regulates autophagic clearance of soluble mutant huntingtin and inhibits aggregation/toxicity in different models of Huntington’s disease

Hegde

Chiki

Petricca³

et al. 2019

Preprint

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Phosphorylation of the N-terminal domain of the Huntingtin (HTT) protein (at T3, S13, and S16) has emerged as a key regulator of HTT stability, clearance, localization, aggregation and toxicity. Herein, we report the discovery and validation of a kinase, TANK-binding kinase 1 (TBK1), that specifically and efficiently phosphorylates both wild-type and mutant full-length or N-terminal fragments of HTT in vitro (S13/S16) and in cell/ neuronal cultures (S13). We show that overexpression of TBK1 in mammalian cells, primary neurons and a Caenorhabditis elegans model of Huntington's Disease (HD) increases mutant HTTex1 phosphorylation, lowers its levels, increases its nuclear localization and significantly reduces its aggregation and cytotoxicity. Our mechanistic studies demonstrate that the TBK1-mediated neuroprotective effects are due to phosphorylation-dependent inhibition of mutant HTTex1 aggregation and an increase in autophagic flux. These findings suggest that upregulation and/or activation of TBK1 represents a viable strategy for the treatment of HD.3

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Section: Resultsmentioning

confidence: 73%

Section: Resultsmentioning

confidence: 99%

Section: Consistent With Our Observation Inmentioning

confidence: 99%

mentioning

confidence: 95%

mentioning

confidence: 99%

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TBK1 regulates autophagic clearance of soluble mutant huntingtin and inhibits aggregation/toxicity in different models of Huntington’s disease

Hegde

Chiki

Petricca³

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

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Role of Post‐translational Modifications in Alzheimer's Disease

2020

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The global burden of Alzheimer's disease (AD) is growing. Valiant efforts to develop clinical candidates for treatment have continuously met with failure. Currently available palliative treatments are temporary and there is a constant need to search for reliable disease pathways, biomarkers and drug targets for developing diagnostic and therapeutic tools to address the unmet medical needs of AD. Challenges in drug‐discovery efforts raise further questions about the strategies of current conventional diagnosis; drug design; and understanding of disease pathways, biomarkers and targets. In this context, post‐translational modifications (PTMs) regulate protein trafficking, function and degradation, and their in‐depth study plays a significant role in the identification of novel biomarkers and drug targets. Aberrant PTMs of disease‐relevant proteins could trigger pathological pathways, leading to disease progression. Advancements in proteomics enable the generation of patterns or signatures of such modifications, and thus, provide a versatile platform to develop biomarkers based on PTMs. In addition, understanding and targeting the aberrant PTMs of various proteins provide viable avenues for addressing AD drug‐discovery challenges. This review highlights numerous PTMs of proteins relevant to AD and provides an overview of their adverse effects on the protein structure, function and aggregation propensity that contribute to the disease pathology. A critical discussion offers suggestions of methods to develop PTM signatures and interfere with aberrant PTMs to develop viable diagnostic and therapeutic interventions in AD.

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Site‐Specific Phosphorylation of Huntingtin Exon 1 Recombinant Proteins Enabled by the Discovery of Novel Kinases

et al. 2020

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Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of exon 1 of the Huntingtin protein (Httex1) play important roles in modulating its cellular properties and functions in health and disease. In particular, phosphorylation of threonine and serine residues (T3, S13, and/or S16) has been shown to inhibit Htt aggregation in vitro and inclusion formation in cellular and animal models of Huntington's disease (HD). In this paper, we describe a new and simple methodology for producing milligram quantities of highly pure wild-type or mutant Httex1 proteins that are site-specifically phosphorylated at T3 or at both S13 and S16. This advance was enabled by 1) the discovery and validation of novel kinases that efficiently phosphorylate Httex1 at S13 and S16 (TBK1), at T3 (GCK) or T3 and S13 (TNIK and HGK), and 2) the development of an efficient methodology for producing recombinant native Httex1 proteins by using a SUMO-fusion expression and purification strategy. [26] As a proof of concept, we demonstrate how this method can be applied to produce Httex1 proteins that are both site-specifically phosphorylated and fluorescently or isotopically labeled. Together, these advances should increase access to these valuable tools and expand the range of methods and experimental approaches that can be used to elucidate the mechanisms by which phosphorylation influences Httex1 or HTT structure, aggregation, interactome, and function(s) in health and disease.

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Ultrasensitive quantitative measurement of huntingtin phosphorylation at residue S13

Cited by 14 publications

References 29 publications

TBK1 regulates autophagic clearance of soluble mutant huntingtin and inhibits aggregation/toxicity in different models of Huntington’s disease

TBK1 regulates autophagic clearance of soluble mutant huntingtin and inhibits aggregation/toxicity in different models of Huntington’s disease

Role of Post‐translational Modifications in Alzheimer's Disease

Site‐Specific Phosphorylation of Huntingtin Exon 1 Recombinant Proteins Enabled by the Discovery of Novel Kinases

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