Nascent mutant Huntingtin exon 1 chains do not stall on ribosomes during translation but aggregates do recruit machinery involved in ribosome quality control

AbstractMutations that cause Huntington’s Disease involve a polyglutamine (polyQ) sequence expansion beyond 35 repeats in exon 1 of Huntingtin. Intracellular inclusion bodies of mutant Huntingtin protein are a key feature of Huntington’s disease brain pathology. We previously showed that in cell culture the formation of inclusions involved the assembly of disordered structures of mHtt exon 1 fragments (Httex1) and they were enriched with translational machinery when first formed. We hypothesized that nascent mutant Httex1 chains co-aggregate during translation by phase separation into liquid-like disordered aggregates and then convert to more rigid, amyloid structures. Here we further examined the mechanisms of inclusion assembly in a human epithelial kidney (AD293) cell culture model and examined whether ribosome quality control machinery previously implicated in stalled ribosomes were involved. We found mHttex1 did not appear to stall translation of its own nascent chain and there was no recruitment of RNA into inclusions. However, proteins involved in translation or ribosome quality control were co-recruited into the inclusions (Ltn1 and Rack1) compared to a protein not anticipated to be involved (NACAD). Furthermore, we observed co-aggregation with other proteins previously identified in inclusions, including Upf-1 and chaperone-like proteins Sgta and Hspb1, which also suppressed aggregation at high co-expression levels. The newly formed inclusions contained immobile mHttex1 molecules which points to the disordered aggregates being mechanically rigid prior to amyloid formation.

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Identification of Full-Length Wild-Type and Mutant Huntingtin Interacting Proteins by Crosslinking Immunoprecipitation in Mice Brain Cortex

Journal of Huntington s Disease ◽

10.3233/jhd-210476 ◽

2021 ◽

pp. 1-13

Author(s):

Karen A. Sap ◽

Arzu Tugce Guler ◽

Aleksandra Bury ◽

Dick Dekkers ◽

Jeroen A.A. Demmers ◽

...

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Brain Cortex ◽

Full Length ◽

Biological Processes ◽

Interacting Proteins ◽

Mutant Huntingtin ◽

Wild Type ◽

Mutant Huntingtin Protein ◽

Mice Brain

Background: Huntington’s disease is a neurodegenerative disorder caused by a CAG expansion in the huntingtin gene, resulting in a polyglutamine expansion in the ubiquitously expressed mutant huntingtin protein. Objective: Here we set out to identify proteins interacting with the full-length wild-type and mutant huntingtin protein in the mice cortex brain region to understand affected biological processes in Huntington’s disease pathology. Methods: Full-length huntingtin with 20 and 140 polyQ repeats were formaldehyde-crosslinked and isolated via their N-terminal Flag-tag from 2-month-old mice brain cortex. Interacting proteins were identified and quantified by label-free liquid chromatography-mass spectrometry (LC-MS/MS). Results: We identified 30 interactors specific for wild-type huntingtin, 14 interactors specific for mutant huntingtin and 14 shared interactors that interacted with both wild-type and mutant huntingtin, including known interactors such as F8a1/Hap40. Syt1, Ykt6, and Snap47, involved in vesicle transport and exocytosis, were among the proteins that interacted specifically with wild-type huntingtin. Various other proteins involved in energy metabolism and mitochondria were also found to associate predominantly with wild-type huntingtin, whereas mutant huntingtin interacted with proteins involved in translation including Mapk3, Eif3h and Eef1a2. Conclusion: Here we identified both shared and specific interactors of wild-type and mutant huntingtin, which are involved in different biological processes including exocytosis, vesicle transport, translation and metabolism. These findings contribute to the understanding of the roles that wild-type and mutant huntingtin play in a variety of cellular processes both in healthy conditions and Huntington’s disease pathology.

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The pathobiology of perturbed mutant huntingtin protein–protein interactions in Huntington's disease

Journal of Neurochemistry ◽

10.1111/jnc.14853 ◽

2019 ◽

Vol 151 (4) ◽

pp. 507-519 ◽

Cited By ~ 10

Author(s):

Erich E. Wanker ◽

Anne Ast ◽

Franziska Schindler ◽

Philipp Trepte ◽

Sigrid Schnoegl

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Protein Interactions ◽

Mutant Huntingtin ◽

Protein Protein Interactions ◽

Huntingtin Protein ◽

Mutant Huntingtin Protein

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Conformational studies of pathogenic expanded polyglutamine protein deposits from Huntington’s disease

Experimental Biology and Medicine ◽

10.1177/1535370219856620 ◽

2019 ◽

Vol 244 (17) ◽

pp. 1584-1595 ◽

Cited By ~ 3

Author(s):

Irina Matlahov ◽

Patrick CA van der Wel

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Structural Biology ◽

Age Of Onset ◽

Repeat Expansion ◽

Cag Repeat ◽

Mutant Huntingtin ◽

Cag Repeat Expansion ◽

Recent Developments ◽

Mutant Huntingtin Protein

Huntington’s disease, like other neurodegenerative diseases, continues to lack an effective cure. Current treatments that address early symptoms ultimately fail Huntington’s disease patients and their families, with the disease typically being fatal within 10–15 years from onset. Huntington’s disease is an inherited disorder with motor and mental impairment, and is associated with the genetic expansion of a CAG codon repeat encoding a polyglutamine-segment-containing protein called huntingtin. These Huntington’s disease mutations cause misfolding and aggregation of fragments of the mutant huntingtin protein, thereby likely contributing to disease toxicity through a combination of gain-of-toxic-function for the misfolded aggregates and a loss of function from sequestration of huntingtin and other proteins. As with other amyloid diseases, the mutant protein forms non-native fibrillar structures, which in Huntington’s disease are found within patients’ neurons. The intracellular deposits are associated with dysregulation of vital processes, and inter-neuronal transport of aggregates may contribute to disease progression. However, a molecular understanding of these aggregates and their detrimental effects has been frustrated by insufficient structural data on the misfolded protein state. In this review, we examine recent developments in the structural biology of polyglutamine-expanded huntingtin fragments, and especially the contributions enabled by advances in solid-state nuclear magnetic resonance spectroscopy. We summarize and discuss our current structural understanding of the huntingtin deposits and how this information furthers our understanding of the misfolding mechanism and disease toxicity mechanisms. Impact statement Many incurable neurodegenerative disorders are associated with, and potentially caused by, the amyloidogenic misfolding and aggregation of proteins. Usually, complex genetic and behavioral factors dictate disease risk and age of onset. Due to its principally mono-genic origin, which strongly predicts the age-of-onset by the extent of CAG repeat expansion, Huntington’s disease (HD) presents a unique opportunity to dissect the underlying disease-causing processes in molecular detail. Yet, until recently, the mutant huntingtin protein with its expanded polyglutamine domain has resisted structural study at the atomic level. We present here a review of recent developments in HD structural biology, facilitated by breakthrough data from solid-state NMR spectroscopy, electron microscopy, and complementary methods. The misfolded structures of the fibrillar proteins inform our mechanistic understanding of the disease-causing molecular processes in HD, other CAG repeat expansion disorders, and, more generally, protein deposition disease.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1705372114 ◽

2017 ◽

Vol 114 (50) ◽

pp. E10809-E10818 ◽

Cited By ~ 26

Author(s):

Cristina Cariulo ◽

Lucia Azzollini ◽

Margherita Verani ◽

Paola Martufi ◽

Roberto Boggio ◽

...

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Posttranslational Modifications ◽

Posttranslational Modification ◽

Protein Conformation ◽

Clinical Samples ◽

Biophysical Properties ◽

Mutant Huntingtin Protein ◽

Exon 1 ◽

Bona Fide

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.

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Disease-related Huntingtin seeding activities in cerebrospinal fluids of Huntington’s disease patients

Scientific Reports ◽

10.1038/s41598-020-77164-1 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

C. Y. Daniel Lee ◽

Nan Wang ◽

Koning Shen ◽

Matthew Stricos ◽

Peter Langfelder ◽

...

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Disease Onset ◽

High Sensitivity ◽

Mutant Huntingtin ◽

Disease Manifestation ◽

Specific Domain ◽

Disease States ◽

Exon 1 ◽

A Cell

AbstractIn Huntington’s disease (HD), the mutant Huntingtin (mHTT) is postulated to mediate template-based aggregation that can propagate across cells. It has been difficult to quantitatively detect such pathological seeding activities in patient biosamples, e.g. cerebrospinal fluids (CSF), and study their correlation with the disease manifestation. Here we developed a cell line expressing a domain-engineered mHTT-exon 1 reporter, which showed remarkably high sensitivity and specificity in detecting mHTT seeding species in HD patient biosamples. We showed that the seeding-competent mHTT species in HD CSF are significantly elevated upon disease onset and with the progression of neuropathological grades. Mechanistically, we showed that mHTT seeding activities in patient CSF could be ameliorated by the overexpression of chaperone DNAJB6 and by antibodies against the polyproline domain of mHTT. Together, our study developed a selective and scalable cell-based tool to investigate mHTT seeding activities in HD CSF, and demonstrated that the CSF mHTT seeding species are significantly associated with certain disease states. This seeding activity can be ameliorated by targeting specific domain or proteostatic pathway of mHTT, providing novel insights into such pathological activities.

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Transcriptional profiles for distinct aggregation states of mutant Huntingtin exon 1 protein unmask new Huntington's disease pathways

Molecular and Cellular Neuroscience ◽

10.1016/j.mcn.2017.07.004 ◽

2017 ◽

Vol 83 ◽

pp. 103-112 ◽

Cited By ~ 9

Author(s):

Nagaraj S. Moily ◽

Angelique R. Ormsby ◽

Aleksandar Stojilovic ◽

Yasmin M. Ramdzan ◽

Jeannine Diesch ◽

...

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Mutant Huntingtin ◽

Transcriptional Profiles ◽

Exon 1 ◽

Disease Pathways

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The huntingtin inclusion is a dynamic phase-separated compartment

Life Science Alliance ◽

10.26508/lsa.201900489 ◽

2019 ◽

Vol 2 (5) ◽

pp. e201900489 ◽

Cited By ~ 5

Author(s):

Fahmida Aktar ◽

Chakkapong Burudpakdee ◽

Mercedes Polanco ◽

Sen Pei ◽

Theresa C Swayne ◽

...

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Vacuolar Membrane ◽

Mutant Huntingtin ◽

Small Particles ◽

Huntingtin Protein ◽

Disordered Protein ◽

Dynamic Phase ◽

Mutant Huntingtin Protein ◽

Polyglutamine Tract

Inclusions of disordered protein are a characteristic feature of most neurodegenerative diseases, including Huntington’s disease. Huntington’s disease is caused by expansion of a polyglutamine tract in the huntingtin protein; mutant huntingtin protein (mHtt) is unstable and accumulates in large intracellular inclusions both in affected individuals and when expressed in eukaryotic cells. Using mHtt-GFP expressed in Saccharomyces cerevisiae, we find that mHtt-GFP inclusions are dynamic, mobile, gel-like structures that concentrate mHtt together with the disaggregase Hsp104. Although inclusions may associate with the vacuolar membrane, the association is reversible and we find that inclusions of mHtt in S. cerevisiae are not taken up by the vacuole or other organelles. Instead, a pulse-chase study using photoconverted mHtt-mEos2 revealed that mHtt is directly and continuously removed from the inclusion body. In addition to mobile inclusions, we also imaged and tracked the movements of small particles of mHtt-GFP and determine that they move randomly. These observations suggest that inclusions may grow through the collision and coalescence of small aggregative particles.

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