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

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.

Ultrasensitive quantitative measurement of huntingtin phosphorylation at residue S13

Sean Michael Deguire, Hilal Lashuel

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.

ACADEMIC PRESS INC ELSEVIER SCIENCE2020

An integrative in silico methodology for the identification of modulators of macrophage migration inhibitory factor (MIF) tautomerase activity

Farah El Turk, Bruno Claude Daniel Fauvet, Hilal Lashuel, Hajer Ouertatani-Sakouhi

Macrophage migration inhibitory factor (MIF) is a major proinflammatory cytokine that has been increasingly implicated in the pathogenesis of several inflammatory, autoimmune, infectious and oncogenic diseases. Accumulating evidence suggests that the tautomerase activity of MIF plays a role in modulating some of its intra- and extra-cellular activities. Therefore, the identification and development of small-molecule inhibitors targeting the catalytic activity of MIF has emerged as an attractive and viable therapeutic strategy to attenuate its function in health and disease. Herein we report a novel virtual screening protocol for the discovery of new inhibitors of MIF's tautomerase activity. Our protocol takes into account the flexibility and dynamics of the catalytic site by coupling molecular dynamics (MD) simulations aimed at modeling the protein's flexibility in solution to (i) docking with FlexX, or (ii) docking with FlexX and pharmacophoric filtering with Unity. In addition, we applied in parallel a standalone docking using the new version of Surflex software. The three approaches were used to screen the ChemBridge chemical library and the inhibitory activity of the top-ranked 333 compound obtained from each approach (1000 compound in total) was assessed in vitro using the tautomerase assay. This biochemical validation process resulted in the identification of 12 novel MIF inhibitors corresponding to a 1.2% hit rate. Six of these hits came from Surflex docking; two from FlexX docking with MD simulations and four hits were identified with MDS and pharmacophore filtering with minimal overlap between the hits from each approach. Six hits were identified with IC50 values lower than 10 microM (three hits with IC50 lower than 1 microM); four were shown to be suicide inhibitors and act via covalent modification of the N-terminal catalytic residues Pro1. One additional inhibitor, N-phenyl-N-1,3,4-thiadiazol-2-yl-thiourea, (IC50=300 nM) was obtained from FlexX docking combined to pharmacophoric filtering on one of the eight MD structures. These results demonstrate the power of integrative in silico approaches in the discovery of new modulator of MIF's tautomerase activity. The chemical diversity and mode of action of these compounds suggest that they could be used as molecular probes to elucidate the functions and biology of MIF and as lead candidates in drug developments of anti-MIF drugs.

Elsevier2010

Huntington's disease modeling and treatment

Diana Zala

Huntington's disease (HD) is a mid-life-onset neurodegenerative disorder characterized by involuntary movements, personality changes and dementia. It progresses to death within 10-20 years after onset. There is currently no cure to treat this fatal disease. In HD patients, the protein huntingtin contains an abnormal expansion of a polyglutamine tract, which leads to the selective death of striatal neurons. The functions of huntingtin, as well as the dysfunctions induced by the mutation are still poorly understood. The first chapter of this thesis describes the state of the art in the study of Huntington's disease: huntingtin (htt), the protein which induces the disease; the hypothesis of toxic pathways induced by the mutant htt and the proposed therapeutic strategies to interfere with them; the available cellular and animal models and finally; the promise of gene therapy for neurodegenerative diseases such as Huntington's disease. The second chapter presents a new cellular model of the developed by infecting primary cultures with lentiviral vectors expressing a mutant htt fragment. This model is characterized by generalized neuronal transgene chronic pathology, expression, neuronal dysfunction and finally cell death. The slow progression in this model allows the study of the cascade of events leading to cell death. These unique characteristics allow the investigation of the pathological events induced by htt expression with analytical techniques on the entire neuronal population. The chapter ends with the description of an experiment showing the neuroprotective effects of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) on thiese neurons. The third chapter describes the delivery of CNTF in transgenic HD mice which express the human full-length htt (YAC72). CNTF was delivered directly to the affected neurons by striatal lentiviral injection. The sustained one-year delivery was not associated with side effects; it was correlated to reduced hyperactivity and a reduction in degenerating neurons. However, a neuroprotective effect was difficult to establish, due to the very mild and subtle pathology occurring in these animals. The fourth chapter considers delivery of BDNF in two genetic rodent HD models: in the mild phenotypic HD mice (YAC128) and a more severe rat model based on the lentiviral delivery of mutant htt in the striatum. No neuroprotective effect of BDNF could be detected in either animal model. The conclusions of this thesis discuss the potential of lentiviral vectors in modeling neurodegenerative diseases in vitro and in vivo, as well as their potential role in the treatment of such diseases. The perspective gained by the developed cellular model toward the understanding of the molecular events induced by mutant huntingtin is discussed, as well as the difficulties in testing neuroprotective approaches on animal models of HD.