Ultrasensitive quantitative measurement of huntingtin phosphorylation at residue S13

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion of a CAG triplet repeat (encoding for a polyglutamine tract) within the first exon of the huntingtin gene. Expression of the mutant huntingtin (mHTT) protein can result in the production of N-terminal fragments with a robust propensity to form oligomers and aggregates, which may be causally associated with HD pathology. Several lines of evidence indicate that N17 phosphorylation or pseudophosphorylation at any of the residues T3, S13 or S16, alone or in combination, modulates mHTT aggregation, subcellular localization and toxicity. Consequently, increasing N17 phosphorylation has been proposed as a potential therapeutic approach. However, developing genetic/pharmacological tools to quantify these phosphorylation events is necessary in order to subsequently develop tool modulators, which is difficult given the transient and incompletely penetrant nature of such post-translational modifications. Here we describe the first ultrasensitive sandwich immunoassay that quantifies HTT phosphorylated at residue S13 and demonstrate its utility for specific analyte detection in preclinical models of HD. (C) 2019 Published by Elsevier Inc.

The Role of Post-translational Modifications on the Energy Landscape of Huntingtin N-Terminus

Charlotte Julie Caroline Gehin, Hilal Lashuel

Huntington disease is a neurodegenerative disease characterized by a polymorphic tract of polyglutamine repeats in exon 1 of the huntingtin protein, which is thought to be responsible for protein aggregation and neuronal death. The polyglutamine tract is preceded by a 17-residue sequence that is intrinsically disordered. This region is subject to phosphorylation, acetylation and other post-translational modifications in vivo, which modulate its secondary structure, aggregation and, subcellular localization. We used Molecular Dynamics simulations with a novel Hamiltonian-replica-exchange-based enhanced sampling method, SWISH, and an optimal combination of water and protein force fields to study the effects of phosphorylation and acetylation as well as cross-talk between these modifications on the huntingtin N-terminus. The simulations, validated by circular dichroism, were used to formulate a mechanism by which the modifications influence helical conformations. Our findings have implications for understanding the structural basis underlying the effect of PTMs in the aggregation and cellular properties of huntingtin.

2019

Development of novel methods and tools to decipher the huntingtin post-translation modifications code

Anass Chiki

Huntington's disease (HD) is a fatal genetic neurodegenerative disorder caused by a CAG repeat expansion in the Huntingtin gene of more than 36 repeats. This repeat is translated into a polyglutamine (polyQ) stretch within the first exon-encoded region of the Huntingtin protein (Httex1). The aggregation of expanded Huntingtin protein (Htt) in the striatum and cortex has been implicated in neuronal dysfunction and death. Recent studies have shown that mutations that mimic post-translational modifications (PTMs) in the first 17 N-terminal amino acids (Nt17) of the protein dramatically influence the aggregation, subcellular localization, and toxicity of Httex1 and full-length Htt proteins. These findings suggest that these modifications may serve as reversible molecular switches for regulating Htt function in health and disease. However, assessing the effect of authentic PTMs and their cross-talk has not been possible due to the lack of knowledge about the enzymes responsible for these modifications. Moreover, the high aggregation propensity of Httex1 with increased polyQ content hampered the development of an efficient method to produce post-translationally modified proteins. By consequence, previous biophysical studies were mostly carried out using tagged Httex1 protein, or model peptides bearing PTM mimetics mutations which do not fully capture the chemical properties of the bona fide PTMs. Therefore, we developed a semisynthetic methodology that permits site-specific modification of the mutant Httex1 (at single or multiple residues). We used this strategy to investigate the effect of acetylation (at K6, K9, or K15) and phosphorylation (at T3), as well as the cross-talk between these modifications on the structure and aggregation of wild-type and mutant Httex1. Our results demonstrated that T3 phosphorylation (pT3) significantly inhibited the aggregation of mutant Httex1 (43Q), whereas only partial inhibition of aggregation was achieved by the phosphomimetic mutation (T3D). Acetylation of single ly-sine residues, K6, K9, or K15 (AcK6, AcK9, or AcK15), did not affect the aggregation of Httex1. Interestingly, AcK6, but not at AcK9 or K15, reversed the inhibitory effect of pT3. Next, we discovered novel kinases GCK and TBK1 that phosphorylate efficiently and specifically T3 and S13/S16, respectively. We exploited these kinases to produce homogenously phosphorylated recombinant Httex1 at T3 or both S13 and S16. We generated, using this method, suitable for structural and cellular applications. Finally, we combined chemical, semisynthetic and enzymatic methods to produce mutant Httex1 proteins bearing oxidized Methionine at position 8 (oxM8) alone or in combination with other Nt17 PTMs (pT3 or AcK6). We ob-served that oxM8 delayed the aggregation of mutant Httex1 and decreased the helical content of the Nt17 independently of the presence of other PTMs. Together, these findings provide novel insight into the role of Nt17 PTMs in regulating the aggregation of Httex1 and suggest that the aggregation and possibly the function (s) of Httex1 are controlled by complex regulatory mechanisms involving cross-talk between different PTMs. The tools presented in this thesis will open new windows of opportunity to decipher the role of Htt PTMs code in the regulation of Htt function in health and disease.

EPFL2020

A New Chemoenzymatic Semisynthetic Approach Provides Insight into the Role of Phosphorylation beyond Exon1 of Huntingtin and Reveals N-Terminal Fragment Length-Dependent Distinct Mechanisms of Aggregation

Rajasekhar Kolla, Hilal Lashuel, Gopinath Pushparathinam, Andreas Reif, Jonathan Jean-Pierre Ricci, Iman Rostami

Huntington’s disease is a neurodegenerative dis- order caused by the expansion of a polyglutamine repeat (>36Q) in the N-terminal domain of the huntingtin protein (Htt), which renders the protein or fragments thereof more prone to aggregate and form inclusions. Although several Htt N-terminal fragments of different lengths have been identified within Htt inclusions, most studies on the mechanisms, sequence, and structural determinants of Htt aggregation have focused on the Httexon1 (Httex1). Herein, we investigated the aggregation properties of mutant N- terminal Htt fragments of various lengths (Htt171, Htt140, and Htt104) in comparison to mutant Httex1 (mHttex1). We also present a new chemoenzymatic semisynthetic strategy that enables site-specific phosphorylation of Htt beyond Httex1. These advances yielded insights into how post-translational modifications (PTMs) and structured domains beyond Httex1 influence aggregation mechanisms, kinetics, and fibril morphology of longer N-terminal Htt fragments. We demonstrate that phosphorylation at T107 significantly slows the aggregation of mHtt171, whereas phosphorylation at T107 and S116 accelerates the aggregation, underscoring the importance of crosstalk between different PTMs. The mHtt171 proteins aggregate via a different mechanism and form oligomers and fibrillar aggregates with morphological properties that are distinct from that of mHttex1. These observations suggest that different N-terminal fragments could have distinct aggregation mechanisms and that a single polyQ-targeting antiaggregation strategy may not effectively inhibit the aggregation of all N-terminal Htt fragments. Finally, our results underscore the need for further studies to investigate the aggregation mechanisms of Htt fragments and how the various fragments interact with each other and influence Htt toxicity and disease progression.

2021

Development of novel methods and tools to decipher the huntingtin post-translation modifications code

Anass Chiki

EPFL2020

Rajasekhar Kolla, Hilal Lashuel, Gopinath Pushparathinam, Andreas Reif, Jonathan Jean-Pierre Ricci, Iman Rostami

2021